The integrated stress response promotes neural stem cell survival under conditions of mitochondrial dysfunction in neurodegeneration.

Impaired mitochondrial function is a hallmark of aging and a major contributor to neurodegenerative diseases. We have shown that disrupted mitochondrial dynamics typically found in aging alters the fate of neural stem cells (NSCs) leading to impairments in learning and memory. At present, little is known regarding the mechanisms by which neural stem and progenitor cells survive and adapt to mitochondrial dysfunction. Using Opa1-inducible knockout as a model of aging and neurodegeneration, we identify a decline in neurogenesis due to impaired stem cell activation and progenitor proliferation, which can be rescued by the mitigation of oxidative stress through hypoxia. Through sc-RNA-seq, we identify the ATF4 pathway as a critical mechanism underlying cellular adaptation to metabolic stress. ATF4 knockdown in Opa1-deficient NSCs accelerates cell death, while the increased expression of ATF4 enhances proliferation and survival. Using a Slc7a11 mutant, an ATF4 target, we show that ATF4-mediated glutathione production plays a critical role in maintaining NSC survival and function under stress conditions. Together, we show that the activation of the integrated stress response (ISR) pathway enables NSCs to adapt to metabolic stress due to mitochondrial dysfunction and metabolic stress and may serve as a therapeutic target to enhance NSC survival and function in aging and neurodegeneration.

Early postnatal defects in neurogenesis in the 3xTg mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia. The hippocampus, which is one of the sites where neural stem cells reside and new neurons are born, exhibits the most significant neuronal loss in AD. A decline in adult neurogenesis has been described in several animal models of AD. However, the age at which this defect first appears remains unknown. To determine at which stage, from birth to adulthood, the neurogenic deficits are found in AD, we used the triple transgenic mouse model of AD (3xTg). We show that defects in neurogenesis are present as early as postnatal stages, well before the onset of any neuropathology or behavioral deficits. We also show that 3xTg mice have significantly fewer neural stem/progenitor cells, with reduced proliferation and decreased numbers of newborn neurons at postnatal stages, consistent with reduced volumes of hippocampal structures. To determine whether there are early changes in the molecular signatures of neural stem/progenitor cells, we perform bulk RNA-seq on cells sorted directly from the hippocampus. We show significant changes in the gene expression profiles at one month of age, including genes of the Notch and Wnt pathways. These findings reveal impairments in neurogenesis very early in the 3xTg AD model, which provides new opportunities for early diagnosis and therapeutic interventions to prevent neurodegeneration in AD.

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

Long-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions.

Direct FACS Isolation of Neural Stem/Progenitor Lineages from the Adult Brain.

Adult neural stem and progenitor cells reside in the neurogenic niche of the adult brain and have tremendous potential in regenerative medicine. Compelling evidence suggests that adult neurogenesis plays an important role in hippocampal memory formation, plasticity, and mood regulation. Understanding the mechanisms that regulate the function of neural stem/progenitor cells within the brain is a critical step for the development of regenerative strategies to maintain or enhance neurological function. A major challenge in studying these cells is the limited cell number of adult neural stem cells, and the significant changes in their properties induced by in vitro culture and expansion. To best understand the regulation of these cells, they must be studied within their niche context. In this chapter, we provide a simplified protocol for the harvest and isolation of neural stem cell lineages directly from the murine brain, to provide input material for single-cell RNA-seq. This approach will elucidate the true transcriptional signatures and activated pathways in neural stem cell lineages, within the context of their niche environment.

Severe asthma treatment patterns: A multicenter observational study in the Gulf region.

Background: While crucial to the assessment and improvement of asthma control, insights on treatment practices in patients with severe diseases across Gulf nations are lacking. This observational study describes the treatment patterns of adolescents and adults with severe asthma across four countries of the Gulf region and evaluates current levels of asthma control; quality of life (QoL); exacerbation frequency; and the application of cellular, protein, and respiratory biomarkers in assessing asthma severity and inflammation. Methods: Patients (aged >12 years, body weight ≥40 kg) with clinician-diagnosed, severe asthma (guided by the 2018 Global Initiative for Asthma definition) were included in this cross-sectional, multicenter, observational study conducted in the four Gulf countries of Kuwait, Oman, Qatar, and the United Arab Emirates. Data on demographics, treatment patterns, and laboratory parameters (blood eosinophil count [BEC], levels of serum immunoglobulin E [IgE], and fractional exhaled nitric oxide [FeNO]) were extracted from the medical records of patients during a 12-month retrospective period and transcribed onto case report forms. At the Enrollment visit, patients assessed their asthma control and QoL with the self-administered Asthma Control Questionnaire (ACQ) and a standardized version of the Asthma Quality of Life Questionnaire (AQLQ(S)), respectively. Results: Among the 243 patients analyzed, (mean [standard deviation (SD)] age, 48.4 [13.9] years; female, 67.5%), the inhaled corticosteroid (ICS)/long-acting ß2 agonist (LABA) combination was the most prescribed asthma medication (n = 240; 98.8%). Most patients were classified as "uncontrolled," (n = 173; 71.2%) and the majority (n = 206; 84.8%) experienced ≥1 exacerbation(s) in the preceding 12 months. The mean (SD) ACQ score was 2.1 (1.2), which indicated uncontrolled asthma, and the mean (SD) total AQLQ(S) score was 4.7 (1.4), suggesting "some limitation" in overall QoL. BECs during the 12-month period were elevated in most patients (>300 cells/µL [n = 183; 41.7%], 150-300 cells/µL [n = 138; 31.4%], <150 cells/µL [n = 118; 26.9%]), suggesting an eosinophilic asthma phenotype, although no standardized threshold by which to define eosinophilia has yet been confirmed. This study revealed that the biomarkers BEC, serum IgE, and FeNO concentrations were obtained inconsistently by the participating centers. Conclusions: Despite recommended ICS/LABA therapy being prescribed to most patients for their severe disease, the majority experienced uncontrolled asthma and exhibited elevated BECs. These findings indicate the need for enhanced treatment strategies to improve and sustain asthma control in the Gulf region.

Altered mitochondrial fusion drives defensive glutathione synthesis in cells able to switch to glycolytic ATP production.

Mitochondria are highly dynamic organelles. Alterations in mitochondrial dynamics are causal or are linked to numerous neurodegenerative, neuromuscular, and metabolic diseases. It is generally thought that cells with altered mitochondrial structure are prone to mitochondrial dysfunction, increased reactive oxygen species generation and widespread oxidative damage. The objective of the current study was to investigate the relationship between mitochondrial dynamics and the master cellular antioxidant, glutathione (GSH). We reveal that mouse embryonic fibroblasts (MEFs) lacking the mitochondrial fusion machinery display elevated levels of GSH, which limits oxidative damage. Moreover, targeted metabolomics and 13C isotopic labeling experiments demonstrate that cells lacking the inner membrane fusion GTPase OPA1 undergo widespread metabolic remodeling altering the balance of citric acid cycle intermediates and ultimately favoring GSH synthesis. Interestingly, the GSH precursor and antioxidant n-acetylcysteine did not increase GSH levels in OPA1 KO cells, suggesting that cysteine is not limiting for GSH production in this context. Post-mitotic neurons were unable to increase GSH production in the absence of OPA1. Finally, the ability to use glycolysis for ATP production was a requirement for GSH accumulation following OPA1 deletion. Thus, our results demonstrate a novel role for mitochondrial fusion in the regulation of GSH synthesis, and suggest that cysteine availability is not limiting for GSH synthesis in conditions of mitochondrial fragmentation. These findings provide a possible explanation for the heightened sensitivity of certain cell types to alterations in mitochondrial dynamics.

Adherence to medication among adult asthma patients in the Middle East and North Africa: results from the ESMAA study.

BACKGROUND: Low levels of adherence to asthma medication is reported in many countries worldwide. Improved knowledge of adherence in the Middle East and North Africa (MENA) is needed to address this major public healthcare burden. OBJECTIVE: Assess the level of adherence in patients attending a routine consultation and the relationship between adherence, patient/disease characteristics, disease control, and quality of life. METHODS: A large-scale cross-sectional epidemiological study was performed on adults suffering from asthma for at least 1 year and without an acute asthma episode within 4 weeks. Adherence was assessed using the MMAS-4 questionnaire©. Predictive factors of adherence were analyzed with logistic regressions. RESULTS: Overall 7203 eligible patients were included in 577 sites. Mean age was 45.4 years (±14.7), 57.2% were female, mean BMI was 28.5 kg/m2 (±6.0), and 11% were active smokers. Good adherence was observed in 23.6% with a country effect (p < 0.001). Higher age, higher SF-8 Mental component score, and high level of control were associated with good adherence (p < 0.001). Patients treated with a fixed combination (ICS + LABA) have better adherence and patients treated with short-acting beta agonist alone have a lower adherence. Good adherence has been noted in 528 uncontrolled patients suggesting the existence of a subgroup difficult to treat and who have severe asthma. CONCLUSIONS: Asthma adherence in the MENA is unsatisfactory with less than one quarter of asthma patients having good adherence. This finding highlights the need to improve access to treatment, ensure better control follow-up and improved education among healthcare providers and patients.

MCL-1Matrix maintains neuronal survival by enhancing mitochondrial integrity and bioenergetic capacity under stress conditions.

Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 Matrix remains poorly understood. Here, we show MCL-1OM and MCL-1Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

Proton-Triggered Fluorescence Switching in Self-Exfoliated Ionic Covalent Organic Nanosheets for Applications in Selective Detection of Anions.

The exfoliation of covalent organic frameworks into covalent organic nanosheets (CONs) not only helps to reduce fluorescence turn-off phenomena but also provides well-exposed active sites for fast response and recovery for various applications. The present work is an example of rational designing of a structure constructed by condensing triaminoguanidinium chloride (TGCl), an intrinsic ionic linker, with a fluorophore, 2, 5-dimethoxyterephthalaldehyde (DA), to produce highly fluorescent self-exfoliable ionic CONs (DATGCl-iCONs). These fluorescent iCONs are able to sense fluoride ions selectively down to the ppb level via the fluorescence turn-off mechanism. A closer look at the quenching mechanism via NMR, zeta potential measurement, lifetime measurement, and density functional theory calculations reveals unique proton-triggered fluorescence switching behavior of newly synthesized DATGCl-iCONs.

Comprehensive evaluation of demographic, socio-economic and other associated risk factors affecting the occurrence of dengue incidence among Colombo and Kandy Districts of Sri Lanka: a cross-sectional study.

BACKGROUND: Comprehensive understanding of risk factors related to socio-economic and demographic status and knowledge, attitudes and practices (KAP) of local communities play a key role in the design and implementation of community-based vector management programmes, along with the identification of gaps in existing control activities. METHODS: A total of 10 Medical Officers of Health (MOH) areas recording high dengue incidence over the last five years were selected from Colombo (n = 5) and Kandy (n = 5) Districts, Sri Lanka. From each MOH area, 200 houses reporting past dengue incidence were selected randomly as test group (n = 1000 for each district) based on the dengue case records available at relevant MOH offices. Information on socio-economic and demographic status and knowledge, attitudes and practices were gathered using an interviewer administered questionnaire. The control group contained 200 households from each MOH area that had not reported any dengue case and the same questionnaire was used for the assessment (n = 1000 for each district). Statistical comparisons between the test and control groups were carried out using the Chi-square test of independence, cluster analysis, analysis of similarities (ANOSIM) and multi-dimensional scaling (MDS) analysis. RESULTS: Significant differences among the test and control groups in terms of basic demographic and socio-economic factors, living standards, knowledge, attitude and practices, were recognized (P < 0.05 at 95% level of confidence). The test group indicated similar risk factors, while the control group also shared more or less similar characteristics as depicted by the findings of cluster analysis and ANOSIM. Findings of the present study highlight the importance of further improvement in community education, motivation and communication gaps, proper coordination and integration of control programmes with relevant entities. Key infrastructural risk factors such as urbanization and waste collection, should be further improved, while vector controlling entities should focus more on the actual conditions represented by the public on knowledge, attitudes and personal protective practices. CONCLUSIONS: The design of flexible and community friendly intervention programmes to ensure the efficacy and sustainability of controlling dengue vectors through community based integrated vector management strategies, is recommended.

Empirical optimization of risk thresholds for dengue: an approach towards entomological management of Aedes mosquitoes based on larval indices in the Kandy District of Sri Lanka.

BACKGROUND: Larval indices such as Premise Index (PI), Breteau Index (BI) and Container Index (CI) are widely used to interpret the density of dengue vectors in surveillance programmes. These indices may be useful for forecasting disease outbreaks in an area. However, use of the values of these indices as alarm signals is rarely considered in control programmes. Therefore, the current study aims to propose threshold values for vector indices based on an empirical modeling approach for the Kandy District of Sri Lanka. METHODS: Monthly vector indices, viz PI, BI and CI, for Aedes aegypti and Aedes albopictus, of four selected dengue high risk Medical Officer of Health (MOH) areas in the Kandy District from January 2010 to August 2017, were used in the study. Gumbel frequency analysis was used to calculate the exceedance probability of quantitative values for each individual larval index within the relevant MOH area, individually and to set up the threshold values for the entomological management of dengue vectors. RESULTS: Among the study MOH areas, Akurana indicated a relatively high density of both Ae. aegypti and Ae. albopictus, while Gangawata Korale MOH area had the lowest. Based on Ae. aegypti, threshold values were defined for Kandy as low risk (BIagp > 1.77), risk (BIagp > 3.23), moderate risk (BIagp > 4.47) and high risk (BIagp > 6.23). In addition, PI > 6.75 was defined as low risk, while PI > 9.43 and PI>12.82 were defined as moderate and high risk, respectively as an average. CONCLUSIONS: Threshold values recommended for Ae. aegypti (primary vector for dengue) along with cut-off values for PI (for Ae. aegypti and Ae. albopictus), could be suggested as indicators for decision making in vector control efforts. This may also facilitate the rational use of financial allocations, technical and human resources for vector control approaches in Sri Lanka in a fruitful manner.

Regulatory Role of Redox Balance in Determination of Neural Precursor Cell Fate.

In 1990s, reports of discovery of a small group of cells capable of proliferation and contribution to formation of new neurons in the central nervous system (CNS) reversed a century-old concept on lack of neurogenesis in the adult mammalian brain. These cells are found in all stages of human life and contribute to normal cellular turnover of the CNS. Therefore, the identity of regulating factors that affect their proliferation and differentiation is a highly noteworthy issue for basic scientists and their clinician counterparts for therapeutic purposes. The cues for such control are embedded in developmental and environmental signaling through a highly regulated tempo-spatial expression of specific transcription factors. Novel findings indicate the importance of reactive oxygen species (ROS) in the regulation of this signaling system. The elusive nature of ROS signaling in many vital processes from cell proliferation to cell death creates a complex literature in this field. Here, we discuss the emerging thoughts on the importance of redox regulation of proliferation and maintenance in mammalian neural stem and progenitor cells under physiological and pathological conditions. The current knowledge on ROS-mediated changes in redox-sensitive proteins that govern the molecular mechanisms in proliferation and differentiation of these cells is reviewed.

Mevalonate Cascade and Small Rho GTPase in Spinal Cord Injury.

The mevalonate pathway has been extensively studied for its involvement in cholesterol synthesis. Inhibition of this pathway using statins (3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors; HMGR inhibitors) is the primarily selected method due to its cholesterol-lowering effect, making statins the most commonly used (86-94%) cholesterol-lowering drugs in adults. This pathway has several other by-products that are affected by statins including GTPase molecules (guanine triphosphate-binding kinases), such as Rho/Rho-associated coiled kinase (ROCK) kinases, that are implicated in other diseases, including those of the central nervous system (CNS). These molecules control several aspects of neural cell life including axonal growth, cellular migration, and cell death, and therefore, are of increasing interest in the field of spinal cord injury (SCI). Limited regeneration capacity of nerve fibers in adult CNS has been considered the main obstacle for finding a SCI cure. Over the past two decades, the identity of inhibitory factors for regeneration has been widely investigated. It is well-established that the Rho/ROCK kinase system is specifically activated by the components of damaged spinal cord tissue, including oligodendrocytes and myelin, as well as extracellular matrix. This has led many groups to hypothesize that statin therapy may in fact enhance the current neurorestorative approaches. In this mini-review, a summary of SCI pathophysiology is discussed and the current literature targeting the regeneration obstacles in SCI are reviewed, with special attention to recent publications of the past decade. In addition, we focus on the current literature involving the use of pharmacological and molecular inhibitors of small GTPase molecules for treatment of neurotrauma. Inhibiting these molecules has been shown to increase neuroprotection, enhance axonal regeneration, and facilitate the implementation of cell replacement therapies. Based upon available literature, the need for clinical trials involving targeted inhibition of GTPase molecules remains strong. Some of these drugs are widely used for other diseases, and therefore re-purposing their application for neurotrauma can be fasttracked. These approaches can potentially modify the inhibitory environment of nervous tissue to allow the spontaneous repair capacity of injured tissue.

Perturbation of redox balance after thioredoxin reductase deficiency interrupts autophagy-lysosomal degradation pathway and enhances cell death in nutritionally stressed SH-SY5Y cells.

Oxidative damage and aggregation of cellular proteins is a hallmark of neuronal cell death after neurotrauma and chronic neurodegenerative conditions. Autophagy and ubiquitin protease system are involved in degradation of protein aggregates, and interruption of their function is linked to apoptotic cell death in these diseases. Oxidative modification of cysteine groups in key molecular proteins has been linked to modification of cellular systems and cell death in these conditions. Glutathione and thioredoxin systems provide reducing protons that can effectively reverse protein modifications and promote cell survival. The central role of Thioredoxin in inhibition of apoptosis is well identified. Additionally, its involvement in initiation of autophagy has been suggested recently. We therefore aimed to investigate the involvement of Thioredoxin system in autophagy-apoptosis processes. A model of serum deprivation in SH-SY5Y was used that is associated with autophagy and apoptosis. Using pharmacological and RNA-editing technology we show that Thioredoxin reductase deficiency in this model enhances oxidative stress and interrupts the early protective autophagy and promotes apoptosis. This was associated with decreased protein-degradation in lysosomes due to altered lysosomal acidification and accumulation of autophagosomes as well as impairment in proteasome pathway. We further confirmed that the extent of oxidative stress is a determining factor in autophagy- apoptosis interplay, as upregulation of cellular reducing capacity by N-acetylcysteine prevented impairment in autophagy and proteasome systems thus promoted cell viability. Our study provides evidence that excessive oxidative stress inhibits protein degradation systems and affects the final stages of autophagy by inhibiting autolysosome maturation: a novel mechanistic link between protein aggregation and conversion of autophagy to apoptosis that can be applicable to neurodegenerative diseases.

Synthesis and activity of proteasome inhibitors.

Among its various catalytic activities, "chymotrypsin-like" activity of proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, we report results from our investigation on a series of peptidomimetic inhibitors.

Japanese encephalitis virus infection alters both neuronal and astrocytic differentiation of neural stem/progenitor cells.

Japanese encephalitis virus (JEV) predominantly infects neurons and causes damage to the central nervous system (CNS). Neural stem/progenitor cells (NSPCs) constitute multi-potent stem cell population in postnatal/adult brain, with capacity to differentiate into neurons, astrocytes or oligodendrocytes. NSPCs are known to play a pivotal role in CNS repair mechanisms during various neurological disorders. Previous studies from our laboratory have shown that JEV infection of NSPCs depletes the stem-cell pool, which may result in impaired repair functions leading to motor and cognitive deficits in survivors. In the present study, we evaluated the effect of JEV infection on differentiation potential of NSPCs isolated from BALB/c mouse pups (Post natal day 7). Results clearly indicated that, JEV infection was more robust in undifferentiated NSPCs as compared to differentiated ones. Further, JEV infected NSPCs showed hampered differentiation and arrested migration in adherent neurosphere cultures. Interestingly, the neuronal differentiation appeared to be more severely affected by JEV as compared to astrocyte differentiation. The transcription factors involved in both neuronal and astrocyte differentiations were significantly decreased upon JEV infection. Overall, results presented in this study comprehensively provide first evidence for JEV induced alteration of neuronal and astrocyte differentiation.

Wake promoting agents: search for next generation modafinil, lessons learned: part III.

In searching for a next generation molecule to the novel wake promoting agent modafinil (compound 1), a series of fluorene-derived wakefulness enhancing agents were developed and evaluated in rat. Extensive pharmacokinetic studies of a potent member of the series (compound 15) revealed that the wake promotion activity of the analog was likely due to an active metabolite (compound 3).

Serendipity in discovery of proteasome inhibitors.

Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported.

Proteasome inhibitors for cancer therapy.

Proteasome, a large multicatalytic proteinase complex that plays an important role in processing of proteins, has been shown to possess multiple catalytic activities. Among its various activities, the 'chymotrypsin-like' activity of proteasome has emerged as the focus of drug discovery efforts in cancer therapy. Herein we report chiral boronate derived novel, potent, selective and cell-permeable peptidomimetic inhibitors 6 and 7 that displayed activity against various rodent and human tumor cell lines (in vitro).

Wake-promoting agents: search for next generation modafinil: part II.

In search of a next generation molecule to modafinil, a novel wake promoting agent, we previously disclosed bi-phenyl derived racemate compound (±)-2 as a new generation of wake-promoting agent. Here we describe the profiles of the individual enantiomers (-)-2 and (+)-2, respectively.