Aspirin as a potential drug repurposing candidate targeting estrogen receptor alpha in breast cancer: a molecular dynamics and in-vitro study.

Estrogen receptor alpha (ERα) is expressed by 70% of breast cancers (BCs). Any deregulation in ERα signaling is crucial for the initiation and progression of BC. Because of development of resistance to anti-estrogenic compounds, repurposing existing drugs is an apt strategy to avoid a long drug-discovery process. Substantial epidemiologic evidence suggests that Aspirin use reduces the risk of different cancers including BC, while its role as an adjuvant or a possible antineoplastic agent in cancer treatment is being investigated. In this study, we attempted to explore possibilities of ERα inhibition by Aspirin which may act through competitive binding to the ligand binding domain (LBD) of ERα. A list of 48 ERα-LBD crystal structures bound with agonists, antagonists, and selective ER modulators (SERMs) was thoroughly analysed to determine interaction patterns specific to each ligand category. Exhaustive docking and 500 ns molecular dynamics (MD) studies were performed on three ERα - Aspirin complexes generated using agonist, antagonist, and SERM-bound crystal structures. Besides, three ERα crystal structures bound to agonist, antagonist, and SERM respectively were also subjected to MD simulations. Aspirin showed good affinity to LBD of ERα. Comparative analyses of binding patterns, conformational changes and molecular interaction profiles from the docking results and MD trajectories suggests that Aspirin was most stable in complex generated using SERM bound crystal structure of ERα and showed interactions with Gly-521, Ala-350, Leu-525 and Thr-347 like SERMs. In addition, in-vitro assays, qPCR, and immunofluorescent assay demonstrated the decline in the expression of ERα in MCF-7 upon treatment with Aspirin. These preliminary bioinformatical and in-vitro findings may form the basis to consider Aspirin as a potential candidate for targeting ERα, especially in tamoxifen-resistant cancers.Communicated by Ramaswamy H. Sarma.

An image analysis approach to characterize micronuclei differences in different subtypes of breast cancer.

BACKGROUND: Micronuclei (MN) have been used as screening, diagnostic and prognostic markers in multiple cancer types, including breast cancer (BC). However, the question that the MN present in all subtypes of BC are similar or different remains unanswered. We thus hypothesized that MN present in different subtypes of BC may differ in their contents which may be visible as differences in their morphologic and morphometric features. This study was thus carried out with the aim to identify the differences between MN morphometry, complexity, and texture in different subtypes of BC, such as estrogen and progesterone receptor-positive (ER+/PR+; MCF-7, T-47D), human epidermal growth factor receptor-positive (Her2 +;SKBR3) and triple-negative BC (TNBC; MDA-MB-231, MDA-MB-468) cell lines (CLs) by ImageJ software. METHODS: For analysis of MN dimensions, MN irregularity, and texture, we used morphometry and two mathematical computer-assisted algorithms, i.e., fractal dimension (FD) and grey level co-occurrence matrix (GLCM) of ImageJ software. RESULTS: MN area and perimeter values showed differences in the size of MN in different subtypes of BC, with the largest MN in TNBC CLs. GLCM parameters (entropy, angular second moment, inverse difference moment, contrast, and correlation) showed highly heterogenous texture in case of TNBC MN as compared to the others. FD analysis also revealed more complexity and irregularity in MN found in TNBC cells. CONCLUSION: The study for the first time showed morphometric, architectural and texture related differences amongst MN present in different subtypes of BC. The above may reflect differences in their composition and contents. Further, these differences may point towards the distinct mechanisms involved in the formation of MN in different subtypes of BC that need to be explored further.

Identifying Challenges and Potential Solutions for Sustainable Kidney Nutrition Care Delivery in Selected Asian Countries.

BACKGROUND: Recent surveys highlight gross workforce shortage of dietitians in global kidney health and significant gaps in renal nutrition care, with disparities greater in low/low-middle income countries. OBJECTIVE: This paper narrates ground experiences gained through the Palm Tocotrienols in Chronic Hemodialysis (PaTCH) project on kidney nutrition care scenarios and some Asian low-to-middle-income countries namely Bangladesh, India, and Malaysia. METHOD: Core PaTCH investigators from 3 universities (USA and Malaysia) were supported by their postgraduate students (n = 17) with capacity skills in kidney nutrition care methodology and processes. This core team, in turn, built capacity for partnering hospitals as countries differed in their ability to deliver dietitian-related activities for dialysis patients. RESULTS: We performed a structural component analyses of PaTCH affiliated and nonaffiliated (Myanmar and Indonesia) countries to identify challenges to kidney nutrition care. Deficits in patient-centered care, empowerment processes and moderating factors to nutrition care optimization characterized country comparisons. Underscoring these factors were some countries lacked trained dietitians whilst for others generalist dietitians or nonclinical nutritionists were providing patient care. Resolution of some challenges in low-to-middle-income countries through coalition networking to facilitate interprofessional collaboration and task sharing is described. CONCLUSIONS: We perceive interprofessional collaboration is the way forward to fill gaps in essential dietitian services and regional-based institutional coalitions will facilitate culture-sensitive capacity in building skills. For the long-term an advanced renal nutrition course such as the Global Renal Internet Course for Dietitians is vital to facilitate sustainable kidney nutrition care.

Touch imprint smear: A prerequisite to obtain better quality and "true" tumor RNA in breast tissues.

BACKGROUND: RNA is the primary genetic material required for various molecular studies. RNA derived from breast tissue has low quality and quantity compared to that extracted from other tissues. Therefore, optimization of techniques for breast tissue RNA extraction is a challenging but essential requirement. METHODS: RNA was extracted from 60 samples of breast cancer after dividing them into 2 groups. Each tissue was divided into 2 halves for RNA extraction and histopathology respectively. In group 2 RNA was extracted after taking touch imprints whereas group1 was not subjected to any such procedure. Concentration and purity of RNA was assessed by using spectrophotometer and 1% agarose gel followed by RT-PCR for 18 S rRNA and CCND1 gene. RESULTS: Based on microscopic observations of imprints, group 2 samples were further subdivided into 2 subgroups. Group 2 A (n = 30) showing tumor in imprint smears were found to yield best concentration of pure RNA (1846.50 ng/µl and 1.92) as compared to group 2B (n = 15) with no malignancy in imprints (102.61 ng/µl and 1.53). The correlation of imprint smears with their corresponding H&E-stained slides further leads to grouping of each group in 2 groups. RT-PCR analyses showed better melting peaks and high relative expression of CCND1 in group 2 A. CONCLUSION: Touch imprints may provide valuable information regarding presence or absence of tumor in tissue samples being used for extraction of genetic material. This approach can be used as easy, cheap and fast strategy to resolve the doubts associated with RNA being truly representative of the tumor.

Pore formation-independent cell death induced by a ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin (ß-PFT). It exhibits potent hemolytic activity against erythrocytes that appears to be a direct outcome of its pore-forming functionality. However, VCC-mediated cell-killing mechanism is more complicated in the case of nucleated mammalian cells. It induces apoptosis in the target nucleated cells, mechanistic details of which are still unclear. Furthermore, it has never been explored whether the ability of VCC to trigger programmed cell death is stringently dependent on its pore-forming activity. Here, we show that VCC can evoke hallmark features of the caspase-dependent apoptotic cell death even in the absence of the pore-forming ability. Our study demonstrates that VCC mutants with abortive pore-forming hemolytic activity can trigger apoptotic cell death responses and cytotoxicity, similar to those elicited by the wild-type toxin. VCC as well as its pore formation-deficient mutants display prominent propensity to translocate to the target cell mitochondria and cause mitochondrial membrane damage. Therefore, our results for the first time reveal that VCC, despite being an archetypical ß-PFT, can kill target nucleated cells independent of its pore-forming functionality. These findings are intriguing for a ß-PFT, whose destination is generally expected to remain limited on the target cell membranes, and whose mode of action is commonly attributed to the membrane-damaging pore-forming ability. Taken together, our study provides critical new insights regarding distinct implications of the two important virulence functionalities of VCC for the V. cholerae pathogenesis process: hemolytic activity for iron acquisition and cytotoxicity for tissue damage by the bacteria.

Protein Energy Wasting in a Cohort of Maintenance Hemodialysis Patients in Dhaka, Bangladesh.

Malnutrition is associated with high rates of mortality among patients with end stage kidney disease (ESKD). There is a paucity of data from Bangladesh, where around 35,000−40,000 people reach ESKD annually. We assessed protein-energy wasting (PEW) amongst 133 patients at a single hemodialysis setting in Dhaka. Patients were 49% male, age 50 ± 13 years, 62% were on twice-weekly hemodialysis. Anthropometric, biochemical, and laboratory evaluations revealed: BMI 24.1 ± 5.2 kg/m2, mid-arm muscle circumference (MAMC) 21.6 ± 3.6 cm, and serum albumin 3.7 ± 0.6 g/dL. Based on published criteria, 18% patients had PEW and for these patients, BMI (19.8 ± 2.4 vs. 25.2 ± 5.2 kg/m2), MAMC (19.4 ± 2.4 vs. 22.2 ± 3.8 cm), serum albumin (3.5 ± 0.7 vs. 3.8 ± 0.5 g/dL), and total cholesterol (135 ± 34 vs. 159 ± 40 mg/dL), were significantly lower as compared to non-PEW patients, while hand grip strength was similar (19.5 ± 7.6 vs. 19.7 ± 7.3 kg). Inflammatory C-reactive protein levels tended to be higher in the PEW group (20.0 ± 34.8 vs. 10.0 ± 13.9 p = 0.065). Lipoprotein analyses revealed PEW patients had significantly lower low density lipoprotein cholesterol (71 ± 29 vs. 88 ± 31 mg/dL, p < 0.05) and plasma triglyceride (132 ± 51 vs. 189 ± 103 mg/dL, p < 0.05), while high density lipoprotein cholesterol was similar. Nutritional assessments using a single 24 h recall were possible from 115 of the patients, but only 66 of these were acceptable reporters. Amongst these, while no major differences were noted between PEW and non-PEW patients, the majority of patients did not meet dietary recommendations for energy, protein, fiber, and several micronutrients (in some cases intakes were 60−90% below recommendations). Malnutrition Inflammation Scores were significantly higher in PEW patients (7.6 ± 3.1 vs. 5.3 ± 2.7 p < 0.004). No discernible differences were apparent in measured parameters between patients on twice- vs. thrice-weekly dialysis. Data from a larger cohort are needed prior to establishing patient-management guidelines for PEW in this population.

Provision of renal-specific nutrition knowledge for changing dietary practice in Bangladeshi hemodialysis patients.

Objective: Studies show that provision of nutrition knowledge help renal patients make informed food choices. This study aimed to evaluate the impact of nutrition knowledge for changing dietary practice among Bangladeshi dialysis patients. Methods: Following development of a renal-specific nutrition booklet, a pilot study was conducted among 50 hemodialysis patients from a single dialysis setting. Demographic, anthropometric, clinical, biochemical, dietary data, and a 10-item MCQ on renal-specific nutrition information were collected before and 3 months after the provision of the booklet. Results: 52% of the participants were male, 54% had twice weekly dialysis, age 53 ± 12 years, and dialysis vintage was 46 ± 25 months. Serum potassium and phosphorous, dietary potassium, phosphorous, and phosphorous to protein ratio were significantly reduced after the provision of the booklet. Additionally, patients consuming >3 meals/day increased to 66% while adherence to renal-specific cooking method and vegetable preference were significantly increased to 70% and 62%, respectively. Conclusion: Provision of knowledge via renal-specific nutrition booklet was able to improve patients' dietary practice and enhance their dietary adherence to renal specific recommendations. Innovation: The booklet was developed using locally available food items in local language and was found beneficial in low-resource settings where overall health care facilities, including nutrition support are limited.

Salmonella Typhimurium Adhesin OmpV Activates Host Immunity To Confer Protection against Systemic and Gastrointestinal Infection in Mice.

Salmonella enterica Typhimurium is a rod-shaped Gram-negative bacterium that mostly enters the human body through contaminated food. It causes a gastrointestinal disorder called salmonellosis in humans and typhoid-like systemic disease in mice. OmpV, an outer membrane protein of S. Typhimurium, helps in adhesion and invasion of bacteria to intestinal epithelial cells and thus plays a vital role in the pathogenesis of S. Typhimurium. In this study, we have shown that intraperitoneal immunization with OmpV is able to induce high IgG production and protection against systemic disease. Further, oral immunization with OmpV-incorporated proteoliposome (OmpV-proteoliposome [PL]) induces production of high IgA antibody levels and protection against gastrointestinal infection. Furthermore, we have shown that OmpV induces Th1 bias in systemic immunization with purified OmpV, but both Th1 and Th2 polarization in oral immunization with OmpV-proteoliposome (PL). Additionally, we have shown that OmpV activates innate immune cells, such as monocytes, macrophages, and intestinal epithelial cells, in a Toll-like receptor 2 (TLR2)-dependent manner. Interestingly, OmpV is recognized by the TLR1/2 heterodimer in monocytes, but by both TLR1/2 and TLR2/6 heterodimers in macrophages and intestinal epithelial cells. Further, downstream signaling involves MyD88, interleukin-1 receptor-associated kinase (IRAK)-1, mitogen-activated protein kinase (MAPK) (both p38 and Jun N-terminal protein kinase (JNK)), and transcription factors NF-κB and AP-1. Due to its ability to efficiently activate both the innate and adaptive immune systems and protective efficacy, OmpV can be a potential vaccine candidate against S. Typhimurium infection. Further, the fact that OmpV can be recognized by both TLR1/2 and TLR2/6 heterodimers increases its potential to act as good adjuvant in other vaccine formulations.

Evaluation of heat stress through delayed sowing on physicochemical and functional characteristics of grains, whole meals and flours of India wheat.

The physicochemical and functional characteristics of grain, meal and flour of timely sown wheat (TSW) and delayed sown wheat (DSW) were compared to see the effects of heat stress (HS). TSW and DSW of different lines were sown as per the approved timings. DSW experienced higher temperature during flowering and had shorter vegetative and maturation period than TSW. Pasting and dough rheological properties were measured using Rapid Visco-Analyser and Farinograph, respectively, while gliadins and glutenins profiling was done by SDS-PAGE. Delayed sowing decreased grain yield and diameter while increased protein and all categories of gliadins and high molecular weight glutenins. DSW showed higher peak viscosity, breakdown-viscosity and dough stability and lower setback viscosity, damaged starch, arabinoxylans and water absorption than TSW. HS in DSW appeared to lower starch synthesis causing proportionate increase in grain hardness and proteins content leading to changes in milling and rheological characteristics.

Correction: Tallman, D.A., et al. Dietary Patterns and Health Outcomes among African American Maintenance Hemodialysis Patients. Nutrients 2020, 12(3), 797.

The authors wish to make the following correction to this paper [...].

Dietary Patterns and Health Outcomes among African American Maintenance Hemodialysis Patients.

The association between dietary patterns and health outcomes, such as quality of life (QOL), in maintenance hemodialysis (MHD) patients with certain racial backgrounds has not been studied in detail. QOL is a powerful outcome measure in which dietary patterns could be a modifying factor. This study is a secondary analysis examining the association between dietary patterns and health outcomes in 101 African American (AA) maintenance hemodialysis (MHD) patients participating in the Palm Tocotrienols in Chronic Hemodialysis (PATCH) study. Quality of life (QOL) was assessed using the Kidney Disease Quality of Life 36-item survey (KDQOL-36™). Blood samples were analyzed for lipids, lipoprotein subfractions, and inflammatory markers. Food intake was measured using six non-consecutive 24-h dietary recalls over 15 months. Implausible energy intake reports were screened out by comparing reported energy intake (rEI) with predicted total energy expenditure (pTEE). Cluster analysis, using the k-means algorithm, identified two distinct dietary patterns in the study population: a high "sugar sweetened beverage" pattern (hiSSB) and a low "sugar sweetened beverage pattern" (loSSB). In the hiSSB group, consumption of SSB accounted for ~28% of energy intake, while SSB represented only 9% of energy intake in the loSSB group. The hiSSB group was characterized by a higher intake of total calories, sugar and percentage of kilocalories from carbohydrates, whereas the percentage of kilocalories from protein and fat was lower. While additional micronutrient intakes differed between groups (vitamin C, zinc, chromium), these were significantly lower than recommended values in the entire cohort. Patients in the hiSSB group presented with lower high-density lipoprotein cholesterol (HDL-C), lower large HDL particles and smaller low density lipoprotein (LDL) particle diameters. Antidepressant usage was significantly higher in the hiSSB group. Patients in the hiSSB group scored lower across all five KDQOL domains and scored significantly lower in the mental composite domain. MHD patients following a hiSSB dietary pattern had smaller dense LDL particles, lower HDL-C, and a lower QOL. Suboptimal intakes of fruits, vegetables, and grains as well as key micronutrients were evident in both patterns.

The health effects of saturated fats - the role of whole foods and dietary patterns.

The effects of saturated fat on cardiovascular health have been studied for more than half a century. While simple public health messages have been to avoid and/or limit their intake, the underlying research is more complex. It is apparent that the effects of saturated fat on health are dependent both on the presence of other nutrients as well as the associated dietary pattern.

Outer membrane protein OmpV mediates Salmonella enterica serovar typhimurium adhesion to intestinal epithelial cells via fibronectin and α1ß1 integrin.

Salmonella typhimurium is an invasive Gram-negative enteric bacterium, which causes salmonellosis, a type of gastroenteritis in humans and typhoid-like symptoms in mice. Upon entering through the contaminated food and water, S. typhimurium adheres, colonises, and invades intestinal epithelial cells (IECs) of the small intestine. In this study, we have shown that upon deletion of the outer membrane protein OmpV, there is a significant decrease in adherence of S. typhimurium to the IECs, indicating that OmpV is an important adhesin of S. typhimurium. Further, our study showed that OmpV binds to the extracellular matrix component fibronectin and signals through α1ß1 integrin receptor on the IECs and OmpV-mediated activation of α1ß1, resulting in the activation of focal adhesion kinase and F-actin modulation. Actin modulation is crucial for bacterial invasion. To the best of our knowledge, this is the first report of an adhesin mediated its effect through integrin in S. typhimurium. Further, we have observed a decrease in pathogenicity in terms of increased LD50 dose, lesser bacterial numbers in stool, and less colonisation of bacteria in different organs of mice infected with Δompv mutant compared with the wild-type bacteria, thus confirming the crucial role of OmpV in the pathogenesis of S. typhimurium.

Lipids, Lipoprotein Distribution and Nutritional Parameters over the Ramadan Period in Hemodialysis Patients.

The period of Ramadan (R) is associated with dramatic changes in eating habits involving extended periods of fasting on a daily basis. The current study assessed whether lipids and lipoproteins were impacted during R in chronic hemodialysis (HD) patients. Forty-five subjects in an outpatient dialysis clinic in Saudi Arabia were evaluated for anthropometric and lipid changes on a monthly basis before, during as well as one and two months after R. In addition to routine biochemical measures, anthropometric assessments including hand-grip strength (HGS), mid-arm muscle circumference (MAMC), plasma lipids and lipoproteins were evaluated. Dietary assessment was carried out using 24 h recalls. Over the course of the study, changes in renal-related parameters (creatinine, albumin, Kt/V) were minor, as were changes in plasma lipids. Large high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs) accounted for the majority of their respective lipoproteins and their proportions did not change over the study period. Mean LDL particle diameters were higher during the R period, but the changes over the study period were small. Calorie intake during R (2139 ± 709 kcal/d) was significantly higher than the value noted two month post-R (1755 ± 424 kcal/d) and this was associated with significant increases in protein (69 ± 24 vs. 60 ± 24 g/d) and fat (97 ± 38, vs. 73 ± 35 g/d), respectively. No changes were noted with respect to HGS and MAMC. These data show that for HD patients, the period of R results in temporal or non-significant effects on plasma lipids, despite changes in nutrient intake.

PRR Function of Innate Immune Receptors in Recognition of Bacteria or Bacterial Ligands.

Recognition of a bacterial attack is the first and the most important step in clearing the bacteria from the body of the host. Towards this, the host innate immune system employs pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), nucleotide-binding leucine-rich repeat-containing receptors (NLRs) and scavenger receptors (SRs) present mostly in innate immune cells. These receptors sense the presence of bacteria and help in spreading the signal to the host, which results in recruitment of other immune cells leading to the elimination of the bacteria from the system. Since their discovery, a lot has been established about these receptors. Their role has been elucidated not only in pathogen recognition but also in eradication of the dead cells from the system. This review is focussed mainly on their role in the bacterial recognition and how these receptors play a role in eliciting an immune response against bacteria in the host.

A 47-year-old returning traveler with shock.

A 47-year-old man with no significant past medical history, originally from Indonesia, was brought to the ED of an urban US medical center after being found collapsed on the sidewalk in respiratory distress and with an altered sensorium. Upon arrival to the ED, he was tachypneic, with increased work of breathing and an oxygen saturation of 88% on 100% nonrebreather mask, so he was immediately intubated. Following intubation, he became profoundly hypotensive, requiring aggressive crystalloid resuscitation and vasopressor support. Broad-spectrum antimicrobials were administered, including ceftriaxone, vancomycin, levofloxacin, and oseltamivir. Further history elicited subsequently from family members revealed that the patient had returned from a 2-week vacation in Indonesia 6 days prior to presentation. According to relatives, he appeared to be in his usual state of health upon his return and was not seen by anyone thereafter, but in the interim he reportedly had an episode of epistaxis, and text messages received from him became progressively more bizarre.

Effects of protein and omega-3 supplementation, provided during regular dialysis sessions, on nutritional and inflammatory indices in hemodialysis patients.

PURPOSE: Malnutrition and chronic inflammation in dialysis patients negatively impacts prognosis. However, intervening to correct this problem (through nutritional supplementation) is often hampered by poor compliance due to both medical and socioeconomic barriers. We have therefore performed a pilot study to investigate the technical feasibility of "directly observed treatment" of nutritional supplementation (protein and omega-3 fatty acids), administered during regular dialysis sessions. Secondary end points included observation of nutritional and inflammatory status of hypoalbuminemic patients undergoing hemodialysis. METHODS: Main inclusion criteria were serum albumin ≤ 3.9 g/dL (3 months prior to the study). Sixty-three eligible patients agreed to participate. Two intervention groups received 30 mL of a liquid protein supplement plus either 2.4 g omega-3 (1800 mg eicosapentaenoic acid + 600 mg docosahexaenoic acid) or a placebo, three times per week after their routine dialysis session for 6 months. Serum albumin, plasma lipids, and other indicators of nutritional and inflammatory status were measured. RESULTS: Directly observed nutritional supplementation resulted in a significant improvement in the low density lipoprotein cholesterol/high density lipoprotein cholesterol ratio in the omega-3 group as compared to the placebo group (P = 0.043). For the omega-3 group, serum albumin was also marginally higher after 6 months as compared to baseline (P = 0.07). The observed increase in C-reactive protein in the placebo group over 6 months was not apparent in the omega-3 group, although there was no significant difference between groups. Nuclear factor kappa B, malnutrition-inflammation score, normalized protein nitrogen appearance, body mass index, and hemoglobin were unaffected by the intervention. CONCLUSION: "Directly observed treatment" with an omega-3 based supplement (as opposed to a pure protein supplement) showed beneficial effects on the lipid profile, and C-reactive protein levels. Further studies using a combination of outpatient and inpatient "directly observed treatment" of omega-3 based supplementation is warranted.

Genetic iron chelation protects against proteasome inhibition-induced dopamine neuron degeneration.

Impairment of the ubiquitin proteasome system (UPS) and iron accumulation in the substantia nigra (SN) have both been implicated in the pathogenesis of Parkinson's disease (PD). We previously reported that chemical iron chelation can protect against proteasome inhibitor lactacystin-induced dopamine (DA) neurodegeneration in vivo. Here, we tested potential neuroprotection via genetic expression of the iron chelator human ferritin heavy chain (H-ferritin). We found that overexpression of H-ferritin in DA neurons significantly reduced lactacystin-induced nigral DA neuron loss and striatal DA depletion. Overexpression of H-ferritin also attenuated elevated levels of total and ferrous iron as well as the divalent metal ion transporter 1 (DMT1) in the SN following lactacystin treatment. In addition, overexpression of H-ferritin alleviated the inhibitory effects of lactacystin on proteasome activity in the nigral tissues. These results suggest that H-ferritin exerts neuroprotection possibly by modulating iron homeostasis and restoring proteasome activity.

Chronic expression of H-ferritin in dopaminergic midbrain neurons results in an age-related expansion of the labile iron pool and subsequent neurodegeneration: implications for Parkinson's disease.

While ferritin elevation within dopaminergic (DA) neurons of the substantia nigra (SN) is protective against neurodegeneration elicited by two toxin models of Parkinson's disease (PD), MPTP and paraquat, in young animals, its prolonged elevation results in a selective age-related neurodegeneration. A similar age-related neurodegeneration has been reported in iron regulatory protein 2-deficient (IRP2 -/-) mice coinciding with increased ferritin levels within degenerating neurons. This has been speculated to be due to subsequent reductions in the labile iron pool (LIP) needed for the synthesis of iron-sulfur-containing enzymes. In order to assess whether LIP reduction is responsible for age-related neurodegeneration in our ferritin transgenics, we examined LIP levels in ferritin-expressing transgenics with increasing age. While LIP levels were reduced within DA SN nerve terminals isolated from young ferritin transgenics compared to wildtype littermate controls, they were found to be increased in older transgenic animals at the age at which selective neurodegeneration is first noted. Furthermore, administration of the bioavailable iron chelator, clioquinol (CQ), to older mice was found to protect against both increased LIP and subsequent dopaminergic neurodegeneration. This suggests that age-related neurodegeneration in these mice is likely due to increased iron availability rather than its reduction. This may have important implications for PD and other related neurodegenerative conditions in which iron and ferritin have been implicated.

A disruption in iron-sulfur center biogenesis via inhibition of mitochondrial dithiol glutaredoxin 2 may contribute to mitochondrial and cellular iron dysregulation in mammalian glutathione-depleted dopaminergic cells: implications for Parkinson's disease.

Parkinson's disease (PD) is characterized by early glutathione depletion in the substantia nigra (SN). Among its various functions in the cell, glutathione acts as a substrate for the mitochondrial enzyme glutaredoxin 2 (Grx2). Grx2 is involved in glutathionylation of protein cysteine sulfhydryl residues in the mitochondria. Although monothiol glutathione-dependent oxidoreductases (Grxs) have previously been demonstrated to be involved in iron-sulfur (Fe-S) center biogenesis, including that in yeast, here we report data suggesting the involvement of mitochondrial Grx2, a dithiol Grx, in iron-sulfur biogenesis in a mammalian dopaminergic cell line. Given that mitochondrial dysfunction and increased cellular iron levels are two important hallmarks of PD, this suggests a novel potential mechanism by which glutathione depletion may affect these processes in dopaminergic neurons. We report that depletion of glutathione as substrate results in a dose-dependent Grx2 inhibition and decreased iron incorporation into a mitochondrial complex I (CI) and aconitase (m-aconitase). Mitochondrial Grx2 inhibition through siRNA results in a corresponding decrease in CI and m-aconitase activities. It also results in significant increases in iron-regulatory protein (IRP) binding, likely as a consequence of conversion of Fe-S-containing cellular aconitase to its non-Fe-S-containing IRP1 form. This is accompanied by increased transferrin receptor, decreased ferritin, and subsequent increases in mitochondrial iron levels. This suggests that glutathione depletion may affect important pathologic cellular events associated with PD through its effects on Grx2 activity and mitochondrial Fe-S biogenesis.