Detecting oxidative stress biomarkers in neurodegenerative disease models and patients

Oxidative stress is prominent in many neurodegenerative diseases. Along with mitochondrial dysfunction and pathological protein aggregation, increased levels of reactive oxygen and nitrogen species, together with impaired antioxidant defense mechanisms, are frequently observed in Alzheimer’s, Parkinson’s, Huntington’s disease and amyotrophic lateral sclerosis. The presence of oxidative stress markers in patients’ plasma and cerebrospinal fluid may aid early disease diagnoses, as well as provide clues regarding the efficacy of experimental disease-modifying therapies in clinical trials. In preclinical animal models, the detection and localization of oxidatively damaged lipids, proteins and nucleic acids helps to identify most vulnerable neuronal populations and brain areas, and elucidate the molecular pathways and the timeline of pathology progression. Here, we describe the protocol for the detection of oxidative stress markers using immunohistochemistry on formaldehyde-fixed, paraffin-embedded tissue sections, applicable to the analysis of postmortem samples and tissues from animal models. In addition, we provide a simple method for the detection of malondialdehyde in tissue lysates and body fluids, which is useful for screening and the identification of tissues and structures in the nervous system which are most affected by oxidative stress. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.Peer reviewe

Determination of optimal parameters of the pulse width modulation of the 4qs transducer for electriс rolling stock

Досліджено режими роботи однофазного 4qs-перетворювача з широтно-імпульсною модуляцією в складі електрорухомого складу змінного струму. Розроблено методу визначення параметрiв ШIМ, при яких реалізується оптимальний за критерiєм мiнiмiзацiї величини реактивної потужності в системі "локомотив – тягова мережа" режим роботи перетворювача. Особливостями запропонованої методи є розділення процесу визначення оптимальних параметрів ШІМ на 2 етапи, що дозволяє видалити з імітаційної моделі непотрiбнi на даному етапі блоки та зменшити сумарний час моделювання. На першому етапі визначаються значення коефіцієнту потужності та струму ланки постійного струму в усьому діапазоні коефiцiєнтiв модуляції та зсуву між мережевим струмом та опорним синусоїдальним сигналом. Далi, з отриманого масиву даних виділяються пари значень параметрів ШІМ, за яких реалізується найвищий коефіцієнт потужності системи "електровоз – тягова мережа", та заносяться до табличною системи завдання параметрів ШІМ. На другому етапi визначається залежності електричних втрат, а, отже, й ККД, та коефіцієнту нелінійних спотворювань мережевого струму вiд тактової частоти перетворювачi. Визначення електричних втрат ґрунтується на обчисленнi енергiї, що була розсiяна протягом 1 с на IGBT-транзисторi та снаберному резисторi в залежностi вiд миттєвих значень струму через них. Для знаходження параметрiв ШIМ за наведеною методою розроблено iмiтацiйну модель 4qs-перетворювача, проведено iдентифiкацiю параметрiв ШIМ перетворювача електровозу для тестової задачi. Визначено, що енергетичнi показники перетворювача залежать нелiнiйно вiд трьох керуючих величин, що є параметрами ШIМ: коефiцiєнту модуляцiї, зсуву мiж мережевим струмом та опорним синусоїдальним сигналом, та тактовою частоти ШIМ. Визначено, що перетворювач з iдентифiкованими параметрами ШIМ забезпечує одиничний коефiцiєнт потужностi тягової мережi при навантаженнi бiльше 10 % вiд номiналь- ного в режимах тяги та рекуперативного гальмування. Отримано залежнiсть електричних втрат перетворювача та коефiцiєнту нелiнiйних спотворень в тяговiй мережi вiд тактової частоти ШIМ. Визначено, що рацiональне значення тактової частоти лежить в iнтервалi 900…2000 Гц, при цьому ККД перетворювача досягає 98…95 %, коефiцiєнт нелiнiйних спотворень складає 12…5 %. Визначено, що виключення з силового кола снаберної ланки може суттєво зменшити сумарнi електричнi втрати. Встановлено, що втрати на паразитних опорах фiльтрiв незначнi, тому їх можна не враховувати в загальному балансi втрат

Biomolecular Release from Alginate-modified Electrode Triggered by Chemical Inputs Processed through a Biocatalytic Cascade – Integration of Biomolecular Computing and Actuation

Biocatalytic cascades involving more than one or two enzyme-catalyzed step are inefficient inside alginate hydrogel prepared on an electrode surface. The problem originates from slow diffusion of intermediate products through the hydrogel from one enzyme to another. However, enzyme activity can be improved by surface immobilization. We demonstrate that a complex cascade of four consecutive biocatalytic reactions can be designed, with the enzymes immobilized in an LBL-assembled polymeric layer at the alginate-modified electrode surface. The product, hydrogen peroxide, then induces dissolution of iron-cross-linked alginate, which results in release process of entrapped biomolecular species, here fluorescently marked oligonucleotides, denoted F-DNA. The enzymatic cascade can be viewed as a biocomputing network of concatenated AND gates, activated by combinations of four chemical input signals, which trigger the release of F-DNA. The reactions, and diffusion/release processes were investigated by means of theoretical modeling. A bottleneck reaction step associated with one of the enzymes was observed. The developed system provides a model for biochemical actuation triggered by a biocomputing network of reactions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimNational Science Foundation, NSF: CBET-1403208Russian Science Foundation, RSF: 17-13-01096This work was supported by National Science Foundation, USA, (award CBET-1403208) and by Russian Science Foundation (project no. 17-13-01096)

Dicer and microRNAs protect adult dopamine neurons

MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (Dicer(HET)) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (Dicer(CKO)). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.Peer reviewe

α-Synuclein Aggregation Inhibitory Prunolides and a Dibrominated β-Carboline Sulfamate from the Ascidian Synoicum prunum

Seven new polyaromatic bis-spiroketal-containing butenolides, the prunolides D–I (4–9) and cis-prunolide C (10), a new dibrominated β-carboline sulfamate named pityriacitrin C (11), alongside the known prunolides A–C (1–3) were isolated from the Australian colonial ascidian Synoicum prunum. The prunolides D–G (4–7) represent the first asymmetrically brominated prunolides, while cis-prunolide C (10) is the first reported with a cis-configuration about the prunolide’s bis-spiroketal core. The prunolides displayed binding activities with the Parkinson’s disease-implicated amyloid protein α-synuclein in a mass spectrometry binding assay, while the prunolides (1–5 and 10) were found to significantly inhibit the aggregation (>89.0%) of α-synuclein in a ThT amyloid dye assay. The prunolides A–C (1–3) were also tested for inhibition of pSyn aggregate formation in a primary embryonic mouse midbrain dopamine neuron model with prunolide B (2) displaying statistically significant inhibitory activity at 0.5 μM. The antiplasmodial and antibacterial activities of the isolates were also examined with prunolide C (3) displaying only weak activity against the 3D7 parasite strain of Plasmodium falciparum. Our findings reported herein suggest that the prunolides could provide a novel scaffold for the exploration of future therapeutics aimed at inhibiting amyloid protein aggregation and the treatment of numerous neurodegenerative diseases.Peer reviewe

Glial cell line-derived neurotrophic factor receptor REarranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo

Background Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. Methods We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. Results BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+)-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. Conclusion The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. (c) 2019 International Parkinson and Movement Disorder SocietyPeer reviewe

Engineered antibody-functionalized porous silicon nanoparticles for therapeutic targeting of pro-survival pathway in endogenous neuroblasts after stroke

Generation of new neurons by utilizing the regenerative potential of adult neural stem cells (NSCs) and neuroblasts is an emerging therapeutic strategy to treat various neurodegenerative diseases, including neuronal loss after stroke. Committed to neuronal lineages, neuroblasts are differentiated from NSCs and have a lower proliferation rate. In stroke the proliferation of the neuroblasts in the neurogenic areas is increased, but the limiting factor for regeneration is the poor survival of migrating neuroblasts. Survival of neuroblasts can be promoted by small molecules; however, new drug delivery methods are needed to specifically target these cells. Herein, to achieve specific targeting, we have engineered biofunctionalized porous silicon nanoparticles (PSi NPs) conjugated with a specific antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). The PSi NPs loaded with a small molecule drug, SC-79, were able to increase the activity of the Akt signaling pathway in doublecortin positive neuroblasts both in cultured cells and in vivo in the rat brain. This study opens up new possibilities to target drug effects to migrating neuroblasts and facilitate differentiation, maturation and survival of developing neurons. The conjugated PSi NPs are a novel tool for future studies to develop new therapeutic strategies aiming at regenerating functional neurocircuitry after stoke

Ret is essential to mediate GDNF’s neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF’s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF’s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD

Engineered antibody-functionalized porous silicon nanoparticles for therapeutic targeting of pro-survival pathway in endogenous neuroblasts after stroke

Generation of new neurons by utilizing the regenerative potential of adult neural stem cells (NSCs) and neuroblasts is an emerging therapeutic strategy to treat various neurodegenerative diseases, including neuronal loss after stroke. Committed to neuronal lineages, neuroblasts are differentiated from NSCs and have a lower proliferation rate. In stroke the proliferation of the neuroblasts in the neurogenic areas is increased, but the limiting factor for regeneration is the poor survival of migrating neuroblasts. Survival of neuroblasts can be promoted by small molecules; however, new drug delivery methods are needed to specifically target these cells. Herein, to achieve specific targeting, we have engineered biofunctionalized porous silicon nanoparticles (PSi NPs) conjugated with a specific antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). The PSi NPs loaded with a small molecule drug, SC-79, were able to increase the activity of the Akt signaling pathway in doublecortin positive neuroblasts both in cultured cells and in vivo in the rat brain. This study opens up new possibilities to target drug effects to migrating neuroblasts and facilitate differentiation, maturation and survival of developing neurons. The conjugated PSi NPs are a novel tool for future studies to develop new therapeutic strategies aiming at regenerating functional neurocircuitry after stoke.Peer reviewe