PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

Effect of emergent nephrostomy on long-term total and split renal function in patients with upper urinary tract obstruction due to pelvic malignant tumors.

PURPOSE: This study aimed to investigate the impact of nephrostomies on the outcome of total renal function (TRF) and split renal function (SRF) in patients with malignant pelvic tumors associated with upper urinary tract obstruction (UUTO). METHODS: Patients with pelvic tumors suffering severe unilateral hydronephrosis treated at our hospital from 2000 to 2022 were included. Data for nephrostomy placement, short- and long-term renal function, and radiological and nuclear imaging studies were collected. The TRF and SRF of patients who underwent nephrostomy were compared to those who did not. RESULTS: Seven patients were included (rhabdomyosarcoma: 5, ovarian germ cell tumor: 1, malignant rhabdoid tumor: 1). Nephrostomies were placed in four, which were successfully managed without severe infections. Estimated glomerular filtration rate (eGFR) was significantly improved at the end of treatment in patients with nephrostomy. In contrast, eGFR in patients who did not undergo nephrostomy was not improved. Nuclear imaging studies (renograms or renal scintigrams) revealed impaired SRF of the affected kidney compared to the contralateral kidney, even in patients whose eGFR was within normal levels. Notably, SRF showed a trend to improve over time in one patient treated with nephrostomy. CONCLUSION: Nephrostomy for UUTO caused by pelvic tumors may improve renal outcome.

Clinical significance of sarcopenia in children with neuroblastic tumors.

PURPOSE: To elucidate the clinical significance of sarcopenia in children with neuroblastic tumors (NTs). METHODS: We conducted a retrospective observational study and analyzed the z-scores for height, body weight, body mass index, and skeletal muscle index (HT-z, BW-z, BMI-z, and SMI-z) along with the clinical characteristics of 36 children with NTs. SMI-z was calculated from 138 computed tomography scans at diagnosis, during treatment, and at follow-up. The International Neuroblastoma Risk Group classification was used to identify high-risk groups. We analyzed the data at diagnosis for prognostic analysis and changes over time after diagnosis in the HT-z, BW-z, BMI-z, and SMI-z groups. RESULTS: Among the four parameters at diagnosis, only SMI-z predicted overall survival (hazard ratio, 0.58; 95% confidence interval, 0.34-0.99). SMI-z, HT-z, and BW-z significantly decreased over time after diagnosis (P < 0.05), while BMI-z did not (P = 0.11). In surviving high-risk NT cases without disease, SMI-z, HT-z, and BW-z significantly decreased over time (P < 0.05), while BMI-z did not (P = 0.43). CONCLUSION: In children with NT, the SMI-z at diagnosis was a significant prognostic factor and decreased during treatment and follow-up along with HT-z and BW-z. Monitoring muscle mass is important because sarcopenia may be associated with growth impairment.

Cellular prion protein is essential for oligomeric amyloid-ß-induced neuronal cell death.

In Alzheimer disease (AD), amyloid-ß (Aß) oligomer is suggested to play a critical role in imitating neurodegeneration, although its pathogenic mechanism remains to be determined. Recently, the cellular prion protein (PrP(C)) has been reported to be an essential co-factor in mediating the neurotoxic effect of Aß oligomer. However, these previous studies focused on the synaptic plasticity in either the presence or the absence of PrP(C) and no study to date has reported whether PrP(C) is required for the neuronal cell death, the most critical element of neurodegeneration in AD. Here, we show that Prnp(-/-) mice are resistant to the neurotoxic effect of Aß oligomer in vivo and in vitro. Furthermore, application of an anti-PrP(C) antibody or PrP(C) peptide prevents Aß oligomer-induced neurotoxicity. These findings are the first to demonstrate that PrP(C) is required for Aß oligomer-induced neuronal cell death, the pathology essential to cognitive loss.

Impaired Aortic Growth in Neuroblastoma Patients After Intensive Treatment.

PURPOSE: This study evaluated the abdominal aortic diameter in high-risk neuroblastoma (NB) patients and the risk of aortic narrowing following intensive treatment. METHODS: We measured the aortic diameter at four specific levels of the abdominal aorta (diaphragmatic crus, celiac axis, and the root of the superior (SMA) and inferior (IMA) mesenteric arteries) on contrast CT scans. The control group consisted of 56 children with non-oncologic disorders, while the NB group included 35 patients with high-risk abdominal NB. We used regression analysis of age and aortic diameter to determine the regression formula for each level in each group and performed intergroup comparisons using t-test. RESULTS: We evaluated a total of 160 contrast-enhanced CTs performed in the 35 eligible cases. The aortic diameter of pretreated NB patients was not significantly different from the controls. After receiving any treatment, the aortic diameter was significantly smaller in the NB group (p < 0.01 each). Patients who underwent radical surgery, particularly gross total resection (n = 26), had smaller aortic diameters at all levels compared to controls (p < 0.01 each). Patients treated with radiotherapy (RT) had smaller aortic diameters than controls. External beam radiotherapy (EBRT) patients (n = 24) had smaller aortic diameters at all levels except the celiac axis (crus, SMA, IMA; p < 0.01 each), and intraoperative radiotherapy (IORT) ± EBRT patients (n = 5) had smaller aortic diameters at all levels (p < 0.01 each). CONCLUSION: Patients with NB may experience impaired development of the abdominal aorta after multimodal therapy, particularly after RT. Close observation and long-term follow-up is essential to monitor for catastrophic vascular complications. LEVEL OF EVIDENCE: LEVEL III.

Establishment of a quantitative assessment model and web-based calculation tool for the skeletal muscle index in children.

BACKGROUND & AIMS: The skeletal muscle index (SMI) is widely used in adults. The reference values for SMI in children are inadequate and require validation in pediatric patients for clinical usefulness. Therefore, this study developed a quantitative assessment model for SMI in children using standard deviation (SD) curves and validated the model's utility and generalizability. METHODS: We examined three compartments of the abdominal skeletal muscle region. SMI was calculated as skeletal muscle area divided by height squared for each compartment (PMI, psoas muscle index; PSMI, paraspinal muscle index; TSMI, total skeletal muscle index). The optimal model was generated using random grouping methods (training and testing), polynomial regression analysis, and the mean squared error evaluation methods. The generated model was validated with previously published SMI data and clinical data of patients with inflammatory bowel disease. RESULTS: The data of 474 children were analyzed. The previously reported SMI reference values overlapped well with our model. In patients with inflammatory bowel disease, the mean (SD) Z-scores for SMI were low in boys (PMI, -1.15 [1.11]; PSMI, -1.31 [1.07]; TSMI, -0.84 [0.91]) and girls (PMI, -1.22 [1.08]; PSMI, -1.44 [1.19]; TSMI, -0.74 [1.16]). Furthermore, SMI was positively correlated with body weight, body mass index, and serum albumin level, a nutritional marker, and negatively correlated with erythrocyte sedimentation rate, an inflammatory marker. CONCLUSION: We established a quantitative assessment model for SMI and validated the model's generalizability and clinical usefulness. We generated an easy-to-use calculation tool for Z-scores from skeletal muscle area obtained from computed tomography images, age, and height information; it has been made publicly available (http://square.umin.ac.jp/ped-muscle-calc/index.html).

Establishment and validation of reference values for abdominal skeletal muscle compartments in children.

BACKGROUND & AIMS: Sarcopenia, resulting from skeletal muscle loss, is a serious concern in children, for whom nutrition and physical activity are essential for growth. In sarcopenia research, the skeletal muscle mass is often obtained from computed tomography (CT) cross-sectional images in both adults and children; however, the method of evaluating skeletal muscle using CT, especially the area range to be measured, has not yet been validated in children. The aim of this study is to establish reference values for three compartments of the abdominal skeletal muscle area (aSMA) obtained from cross-sectional CT images, and to validate the differences among them by assessing correlations with physical development. METHODS: We conducted a single-centre, retrospective, cross-sectional study and included patients aged 1-17 years who underwent abdominal CT at Chiba University Hospital from 2007 to 2020. Patients with chronic diseases such as malignant tumours and inflammatory bowel disease were excluded from the analysis. aSMA was obtained from CT cross-sections at the lumbar L3-L4 intervertebral disc level by dividing it into three compartments: the psoas muscle area (PMA), paraspinal muscle area (PSMA), and total skeletal muscle area (TSMA). Quartile regression curves by sex and muscle compartment were generated using non-crossover and nonparametric regression quantile estimation. The correlation between each compartment of aSMA with height and weight was assessed using Spearman's rank correlation coefficient. RESULTS: We analysed the abdominal CT images of 593 children (male: n = 335, female: n = 258) and generated curves predictive for the 5th, 25th, 50th, 75th, and 95th percentiles for each compartment of aSMA by sex. In patients aged 13 years and older, boys had significantly larger aSMA areas than girls in all three compartments. Among the three compartments, PSMA had the strongest correlation coefficient with height and weight for both boys and girls. CONCLUSIONS: We generated quantile regression curves for three aSMA compartments obtained from cross-sectional CT images and established reference values in children. PSMA values were most strongly correlated with height and weight among the three aSMA compartments.

High-fat diet-induced obesity and insulin resistance were ameliorated via enhanced fecal bile acid excretion in tumor necrosis factor-alpha receptor knockout mice.

Tumor necrosis factor-α (TNF-α) is one of the main mediators of inflammatory response activated by fatty acids in obesity, and this signaling through TNF-α receptor (TNFR) is responsible for obesity-associated insulin resistance. Recently, TNF-α has shown to affect lipid metabolism including the regulation of lipase activity and bile acid synthesis. However, there is scanty in vivo evidence for the involvement of TNF-α in this process, and the mechanistic role of TNFR remains unclear. In this study, TNFR2 knockout mice (R2KO) and wild-type (WT) mice were fed commercial normal diet (ND) or high-fat diet (HFD) for 8 weeks. In R2KO/HFD mice, the increase in body weight and the accumulation of fat were significantly ameliorated compared with WT/HFD mice in association with the decrease in plasma total cholesterol (137.7±3.1 vs. 98.6±3.1 mg/dL, P<0.005), glucose (221.9±14.7 vs. 167.3±8.1 mg/dL, P<0.01), and insulin (5.1±0.3 vs. 3.4±0.3 ng/mL, P<0.05). Fecal excretion of lipid contents was significantly increased in R2KO mice. In R2KO/HFD mice, the decrease in hepatic cholesterol-7a-hydroxylase activity, the rate-limiting enzyme in bile acid synthesis, was inhibited (1.7±0.2 vs. 8.1±1.0 pmol/min/mg protein, P<0.01). These results suggested that HFD-induced obesity with metabolic derangements could be ameliorated in mice lacking TNF-α receptor 2 via increasing fecal bile acid and lipid content excretion. Therefore, TNF-α signaling through TNFR2 is essentially involved in the bile acid synthesis and excretion of lipids, resulting in its beneficial effects.

In vivo imaging of mitochondrial function in methamphetamine-treated rats.

Abuse of the powerfully addictive psychostimulant, methamphetamine, occurs worldwide. Recent studies have suggested that methamphetamine-induced dopaminergic neurotoxicity is related to oxidative stress. In response to nerve activation, the mitochondrial respiratory chain is rapidly activated. The enhancement of mitochondrial respiratory chain activation may induce oxidative stress in the brain. However, there is little experimental evidence regarding the mitochondrial function after methamphetamine administration in vivo. Here, we evaluated whether a single administration of methamphetamine induces ATP consumption and overactivation of mitochondria. We measured mitochondrial function in two different ways: by monitoring oxygen partial pressure using an oxygen-selective electrode, and by imaging of redox reactions using a nitroxyl radical (i.e., nitroxide) coupled with Overhauser-enhanced magnetic resonance imaging (OMRI). A single administration of methamphetamine to Wistar rats induced dopaminergic nerve activation, ATP consumption and an increase in mitochondrial respiratory chain function in both the striatum and cortex. Furthermore, antioxidant TEMPOL prevented the increase in mitochondrial oxidative damage and methamphetamine-induced sensitization. These findings suggest that energy-supplying reactions after dopaminergic nerve activation are associated with oxidative stress in both the striatum and cortex, leading to abnormal behavior.

Novel purine-based fluoroaryl-1,2,3-triazoles as neuroprotecting agents: synthesis, neuronal cell culture investigations, and CDK5 docking studies.

A series of novel purine-based fluoroaryl triazoles were synthesized using the Cu(I) catalyzed 1,3-dipolar cycloaddition reactions (click reactions), and assayed for their neuroprotective effects using fluorescence electron microscopy. Among these triazoles, o-fluorophenylmetyl-triazole, 7, has comparable neuroprotective effect as that of Flavopiridol (1) and Roscovitine (2), the state of the art CDK inhibitors, against the Aß induced neurotoxicity. These results are substantiated using computer docking methods (DarwinDock/GenDock), which predict that Roscovitine and the triazole 7 bind to the ATP-binding site of CDK5/p25 with comparable binding energies, whereas the corresponding pentafluorophenylmethyl-triazole, 9, has dramatically reduced binding energy (in accordance with its lack of neuroprotection). These combined experimental and theoretical studies support the involvement of CDK5/p25 in the neuronal cell cycle re-entry.

Mislocalization of CDK11/PITSLRE, a regulator of the G2/M phase of the cell cycle, in Alzheimer disease.

Post-mitotic neurons are typically terminally differentiated and in a quiescent status. However, in Alzheimer disease (AD), many neurons display ectopic re-expression of cell cycle-related proteins. Cyclin-dependent kinase 11 (CDK11) mRNA produces a 110-kDa protein (CDK11(p110)) throughout the cell cycle, a 58-kDa protein (CDK11(p58)) that is specifically translated from an internal ribosome entry site and expressed only in the G(2)/M phase of the cell cycle, and a 46-kDa protein (CDK11(p46)) that is considered to be apoptosis specific. CDK11 is required for sister chromatid cohesion and the completion of mitosis. In this study, we found that the expression patterns of CDK11 vary such that cytoplasmic CDK11 is increased in AD cellular processes, compared to a pronounced nuclear expression pattern in most controls. We also investigated the effect of amyloid precursor protein (APP) on CDK11 expression in vitro by using M17 cells overexpressing wild-type APP and APP Swedish mutant phenotype and found increased CDK11 expression compared to empty vector. In addition, amyloid-ß(25-35) resulted in increased CDK11 in M17 cells. These data suggest that CDK11 may play a vital role in cell cycle re-entry in AD neurons in an APP-dependent manner, thus presenting an intriguing novel function of the APP signaling pathway in AD.

Recurrent massive bleeding from a small intestinal arteriovenous malformation after surgery for biliary atresia in an infant: a case report and literature review.

BACKGROUND: Small intestinal arteriovenous malformation (AVM) can cause bleeding. Most small intestinal AVMs occur during adulthood, rarely in infancy. We report a case of an infant with hemorrhage due to small intestinal AVM early and recurrently after Kasai portoenterostomy (PE) for biliary atresia (BA). CASE PRESENTATION: A 51-day-old male infant was admitted to our institution for obstructive jaundice. Laparotomic cholangiography revealed BA (IIIb1µ), and Kasai PE was performed at 60 days of age. On postoperative day 17, he developed massive melena and severe anemia. Contrast-enhanced computed tomography (CT) revealed that the jejunum around the PE site was strongly enhanced with enhancing nodules in the arterial phase, and a wide area of the Roux limb wall was slightly enhanced in the venous phase. As melena continued, emergency laparotomy was performed. There were no abnormal macroscopic findings at the PE site except for a clot in the Roux limb 5 cm away from the PE site, and the Roux limb was resected 5 cm. On further investigation, a red spot was detected on the jejunal serosa 30 cm away from the Roux-en-Y anastomosis site. PE and wedge resection for the red spot were performed. Histopathologically, both specimens indicated AVM. He was jaundice-free 65 days after the first surgery. However, at 7 months of age, he developed massive melena again. Contrast-enhanced CT and upper gastrointestinal endoscopy revealed no bleeding lesions. Hemorrhagic scintigraphy showed a slight accumulation at the hepatic hilum prompting an emergency surgery. Intraoperative endoscopy detected a bleeding lesion at the PE site, and the Roux limb was resected (approximately 6 cm). Intraoperative frozen section analysis of the stump of the resected jejunum revealed no abnormal vessels. PE was performed, and permanent section analysis revealed an AVM in the resected jejunum. The postoperative course was uneventful without re-bleeding. CONCLUSIONS: We experienced a case of recurrent massive bleeding from small intestinal AVM in an infant after surgery for BA. Intraoperative endoscopy and frozen section analysis helped identify the bleeding lesion and perform a complete resection of the small intestinal AVM, even after surgery, in the infant.

Pathological implications of cell cycle re-entry in Alzheimer disease.

The complex neurodegeneration underlying Alzheimer disease (AD), although incompletely understood, is characterised by an aberrant re-entry into the cell cycle in neurons. Pathological evidence, in the form of cell cycle markers and regulatory proteins, suggests that cell cycle re-entry is an early event in AD, which precedes the formation of amyloid-beta plaques and neurofibrillary tangles (NFTs). Although the exact mechanisms that induce and mediate these cell cycle events in AD are not clear, significant advances have been made in further understanding the pathological role of cell cycle re-entry in AD. Importantly, recent studies indicate that cell cycle re-entry is not a consequence, but rather a cause, of neurodegeneration, suggesting that targeting of cell cycle re-entry may provide an opportunity for therapeutic intervention. Moreover, multiple inducers of cell cycle re-entry and their interactions in AD have been proposed. Here, we review the most recent advances in understanding the pathological implications of cell cycle re-entry in AD.

Cellular prion protein and Alzheimer disease: link to oligomeric amyloid-ß and neuronal cell death.

Soluble oligomeric amyloid-ß (Aß) has been suggested to impair synaptic and neuronal function, leading to neurodegeneration that is clinically observed as the memory and cognitive dysfunction characteristic of Alzheimer disease, while the precise mechanism(s) whereby oligomeric Aß causes neurotoxicity remains unknown. Recently, the cellular prion protein (PrP (C) ) was reported to be an essential co-factor in mediating the neurotoxic effect of oligomeric Aß. Our recent study showed that Prnp (-/-) mice are resistant to the neurotoxic effect of oligomeric Aß in vivo and in vitro. Furthermore, application of an anti-PrP (C) antibody or PrP (C) peptide was able to block oligomeric Aß-induced neurotoxicity. These findings demonstrate that PrP (C) may be involved in neuropathologic conditions other than conventional prion diseases, i.e., Creutzfeldt-Jakob disease.

Early induction of c-Myc is associated with neuronal cell death.

Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-ß peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases.

Determination of reactive oxygen species associated with the degeneration of dopaminergic neurons during dopamine metabolism.

Oxidative stress is believed to be an important mechanism underlying dopamine-induced neuronal damage. This study provides X-band electron spin resonance (ESR) spectroscopic evidence for reactive oxygen species (ROS) generation during dopamine metabolism. The authors induced excess dopamine metabolism in the mouse striatum by bathing it in tyramine-containing perfusate using microdialysis. The addition of tyramine to the perfusate raised the levels of extracellular dopamine and hydrogen peroxide significantly. The ESR signal from hydroxy-TEMPO decayed during tyramine perfusion and treatment with a monoamine-oxidase inhibitor or radical scavenger suppressed the signal decay. Decreases in the number of tyrosine hydroxylase-immunopositive fibres and in dopamine concentration after tyramine perfusion were observed. Moreover, the tyramine-perfused mice showed a marked methamphetamine-induced rotational response. Notably, these effects of tyramine were suppressed by the simultaneous perfusion of hydroxy-TEMPO. These findings indicate that the ROS generation, which was monitored by hydroxy-TEMPO, caused oxidative damage to the dopaminergic neurons.

Nanoparticle-chelator conjugates as inhibitors of amyloid-beta aggregation and neurotoxicity: a novel therapeutic approach for Alzheimer disease.

Oxidative stress and amyloid-beta are considered major etiological and pathological factors in the initiation and promotion of neurodegeneration in Alzheimer disease (AD). Insomuch as causes of such oxidative stress, transition metals, such as iron and copper, which are found in high concentrations in the brains of AD patients and accumulate specifically in the pathological lesions, are viewed as key contributors to the altered redox state. Likewise, the aggregation and toxicity of amyloid-beta is dependent upon transition metals. As such, chelating agents that selectively bind to and remove and/or "redox silence" transition metals have long been considered as attractive therapies for AD. However, the blood-brain barrier and neurotoxicity of many traditional metal chelators has limited their utility in AD or other neurodegenerative disorders. To circumvent this, we previously suggested that nanoparticles conjugated to iron chelators may have the potential to deliver chelators into the brain and overcome such issues as chelator bioavailability and toxic side-effects. In this study, we synthesized a prototype nanoparticle-chelator conjugate (Nano-N2PY) and demonstrated its ability to protect human cortical neurons from amyloid-beta-associated oxidative toxicity. Furthermore, Nano-N2PY nanoparticle-chelator conjugates effectively inhibited amyloid-beta aggregate formation. Overall, this study indicates that Nano-N2PY, or other nanoparticles conjugated to metal chelators, may provide a novel therapeutic strategy for AD and other neurodegenerative diseases associated with excess transition metals.

Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study.

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical ((*)OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with (*)OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary (*)OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and (*)OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary (*)OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the (*)OH sources in pathological processes such as Parkinson's disease.