ABSTRACT
Due to the favorable features obtained through the incorporation of fluorine atom(s), fluorinated drugs are a group with emerging pharmaceutical importance. As their commercial availability is still very limited, to expand the range of possible candidates, new fluorinated tryptophan analogs were synthesized. Control of enantiopurity during the synthesis procedure requires that highly efficient enantioseparation methods be available. In this work, the enantioseparation of seven fluorinated tryptophans and tryptophan was studied and compared systematically to (i) develop analytical methods for enantioselective separations and (ii) explore the chromatographic features of the fluorotrytophans. For enantioresolution, macrocyclic glycopeptide-based selectors linked to core-shell particles were utilized, applying liquid chromatography-based methods. Application of the polar-ionic mode resulted in asymmetric and broadened peaks, while reversed-phase conditions, together with mobile-phase additives, resulted in baseline separation for all studied fluorinated tryptophans. The marked differences observed between the methanol and acetonitrile-containing eluent systems can be explained by the different solvation abilities of the bulk solvents of the applied mobile phases. Among the studied chiral selectors, teicoplanin and teicoplanin aglycone were found to work effectively. Under optimized conditions, baseline separations were achieved within 6 min. Ionic interactions were semi-quantitatively characterized and found to not influence enantiorecognition. Interestingly, fluorination of the analytes does not lead to marked changes in the chromatographic characteristics of the methanol-containing eluents, while larger differences were noticed when the polar but aprotic acetonitrile was applied. Experiments conducted on the influence of the separation temperature indicated that the separations are enthalpically driven, with only one exception. Enantiomeric elution order was found to be constant on both teicoplanin and teicoplanin aglycone-based chiral stationary phases (L < D) under all applied chromatographic conditions.
Subject(s)
Glycopeptides , Halogenation , Teicoplanin , Tryptophan , Tryptophan/chemistry , Tryptophan/analogs & derivatives , Glycopeptides/chemistry , Stereoisomerism , Teicoplanin/chemistry , Teicoplanin/analogs & derivatives , Chromatography, Liquid/methods , Chromatography, High Pressure Liquid/methods , Macrocyclic Compounds/chemistryABSTRACT
The prevalence of chronic kidney disease (CKD) is increasing globally, especially in elderly patients. Uremic cardiomyopathy is a common cardiovascular complication of CKD, characterized by left ventricular hypertrophy (LVH), diastolic dysfunction, and fibrosis. Kisspeptins and their receptor, KISS1R, exert a pivotal influence on kidney pathophysiology and modulate age-related pathologies across various organ systems. KISS1R agonists, including kisspeptin-13 (KP-13), hold promise as novel therapeutic agents within age-related biological processes and kidney-related disorders. Our investigation aimed to elucidate the impact of KP-13 on the trajectory of CKD and uremic cardiomyopathy. Male Wistar rats (300-350 g) were randomized into four groups: (I) sham-operated, (II) 5/6 nephrectomy-induced CKD, (III) CKD subjected to a low dose of KP-13 (intraperitoneal 13 µg/day), and (IV) CKD treated with a higher KP-13 dose (intraperitoneal 26 µg/day). Treatments were administered daily from week 3 for 10 days. After 13 weeks, KP-13 increased systemic blood pressure, accentuating diastolic dysfunction's echocardiographic indicators and intensifying CKD-associated markers such as serum urea levels, glomerular hypertrophy, and tubular dilation. Notably, KP-13 did not exacerbate circulatory uremic toxin levels, renal inflammation, or fibrosis markers. In contrast, the higher KP-13 dose correlated with reduced posterior and anterior wall thickness, coupled with diminished cardiomyocyte cross-sectional areas and concurrent elevation of inflammatory (Il6, Tnf), fibrosis (Col1), and apoptosis markers (Bax/Bcl2) relative to the CKD group. In summary, KP-13's influence on CKD and uremic cardiomyopathy encompassed heightened blood pressure and potentially activated inflammatory and apoptotic pathways in the left ventricle.
Subject(s)
Cardiomyopathies , Hypertension , Renal Insufficiency, Chronic , Humans , Rats , Animals , Male , Aged , Kisspeptins , Receptors, Kisspeptin-1 , Rats, Wistar , Renal Insufficiency, Chronic/complications , Cardiomyopathies/complications , Hypertension/complications , FibrosisABSTRACT
Neurogenesis plays a crucial role in cognitive processes. During aging and in Alzheimer's disease (AD), altered neurogenesis and neuroinflammation are evident both in C57BL/6J, APPSwe/PS1dE9 (Tg) mice and humans. AD pathology may slow down upon drug treatment, for example, in a previous study of our group P33, a putative neuroprotective agent was found to exert advantageous effects on the elevated levels of APP, Aß, and neuroinflammation. In the present study, we aimed to examine longitudinal alterations in neurogenesis, neuroinflammation and AD pathology in a transgenic (Tg) mouse model, and assessed the putative beneficial effects of long-term P33 treatment on AD-specific neurological alterations. Hippocampal cell proliferation and differentiation were significantly reduced between 8 and 12 months of age. Regarding neuroinflammation, significantly elevated astrogliosis and microglial activation were observed in 6- to 7-month-old Tg animals. The amounts of the molecules involved in the amyloidogenic pathway were altered from 4 months of age in Tg animals. P33-treatment led to significantly increased neurogenesis in 9-month-old animals. Our data support the hypothesis that altered neurogenesis may be a consequence of AD pathology. Based on our findings in the transgenic animal model, early pharmacological treatment before the manifestation of AD symptoms might ameliorate neurological decline.
Subject(s)
Alzheimer Disease , Neuroprotective Agents , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/pharmacology , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Disease Models, Animal , Humans , Infant , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurogenesis , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Presenilin-1/genetics , Presenilin-1/metabolismABSTRACT
Alzheimer's disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-ß-(Aß-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aß-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aß1-42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.
Subject(s)
Alzheimer Disease , Receptors, sigma , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Disease Models, Animal , Mice , N,N-Dimethyltryptamine , Neurogenesis , Neuroinflammatory Diseases , Peptide Fragments , Receptors, sigma/metabolism , Sigma-1 ReceptorABSTRACT
Neurodegeneration-related human-type beta-amyloid 1-42 aggregates (H-Aß) are one of the biochemical markers and executive molecules in Alzheimer's disease. The exogenic rotifer-specific biopolymer, namely Rotimer, has a protective effect against H-Aß toxicity on Euchlanis dilatata and Lecane bulla monogonant rotifers. Due to the external particle-dependent secreting activity of these animals, this natural exudate exists in a bound form on the surface of epoxy-metal beads, named as Rotimer Inductor Conglomerate (RIC). In this current work the experiential in vitro molecular interactions between Rotimer and Aßs are presented. The RIC form was uniformly used against H-Aß aggregation processes in stagogram- and fluorescent-based experiments. These well-known cell-toxic aggregates stably and quickly (only taking a few minutes) bind to RIC. The epoxy beads (as carriers) alone or the scrambled version of H-Aß (with random amino acid sequence) were the ineffective and inactive negative controls of this experimental system. The RIC has significant interacting, anti-aggregating and disaggregating effects on H-Aß. To detect these experiments, Bis-ANS and Thioflavin T were applied during amyloid binding, two aggregation-specific functional fluorescent dyes with different molecular characteristics. This newly described empirical interaction of Rotimer with H-Aß is a potential starting point and source of innovation concerning targeted human- and pharmaceutical applications.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Alzheimer Disease/metabolism , Amyloid/chemistry , Amyloid beta-Peptides/metabolism , Animals , Biopolymers/pharmacology , Fluorescent Dyes/pharmacology , HumansABSTRACT
In vertebrates, gonadotropin-releasing hormone (GnRH) peptide is the central mediator of reproduction. Homologous peptides have previously also been identified in molluscan species. However, emerging evidence suggests that these molecules might serve diverse regulatory functions and proposes to consider them as corazonin (CRZ). We previously isolated the full-length cDNA of the invGnRH/CRZ peptide (termed ly-GnRH/CRZ) in the well-established invertebrate model species, the great pond snail Lymnaea stagnalis; however, its predicted functions remain to be verified. In this study, we first confirmed the presence of the deduced active peptide from the central nervous system of L. stagnalis. Further, we performed in vivo and in vitro studies to explore the functions of ly-GnRH/CRZ. Injection of sexually mature specimens with synthetic active peptide had an inhibitory effect on locomotion and an acceleratory effect on egg-laying, but had no effect on feeding. The previously predicted modulatory effect of ly-GnRH/CRZ was supported by its identified co-localization with serotonin on the surface of the heart atria. Lastly, we demonstrated not only the presence of ly-GnRH/CRZ in the penial complex but also that ly-GnRH/CRZ-containing neurons project to the efferent penis nerve, suggesting ly-GnRH/CRZ may directly modulate the motor output of this peripheral tissue. Overall, our findings strongly support that ly-GnRH/CRZ is a multifunctional neuropeptide. These results contribute to the understanding of the GnRH superfamily and, more broadly, disciplines such as comparative endocrinology and neurobiology.
Subject(s)
Lymnaea/physiology , Neuropeptides/physiology , Animals , Biological Evolution , Central Nervous System/metabolism , Feeding Behavior , Gonadotropin-Releasing Hormone/chemistry , Gonadotropin-Releasing Hormone/physiology , Locomotion , Lymnaea/chemistry , Neuropeptides/chemistry , OviparityABSTRACT
Kisspeptins (Kp) are RF-amide neuropeptide regulators of the reproductive axis that also influence anxiety, locomotion, and metabolism. We aimed to investigate the effects of intracerebroventricular Kp-8 (an N-terminally truncated octapeptide) treatment in Wistar rats. Elevated plus maze (EPM), computerized open field (OF), and marble burying (MB) tests were performed for the assessment of behavior. Serum LH and corticosterone levels were determined to assess kisspeptin1 receptor (Kiss1r) activation and hypothalamic-pituitary-adrenal axis (HPA) stimulation, respectively. GABA release from the nucleus accumbens (NAc) and dopamine release from the ventral tegmental area (VTA) and NAc were measured via ex vivo superfusion. Kp-8 decreased open arm time and entries in EPM, and also raised corticosterone concentration, pointing to an anxiogenic effect. Moreover, the decrease in arm entries in EPM, the delayed increase in immobility accompanied by reduced ambulatory activity in OF, and the reduction in interactions with marbles show that Kp-8 suppressed exploratory and spontaneous locomotion. The increase in GABA release from the NAc might be in the background of hypolocomotion by inhibiting the VTA-NAc dopaminergic circuitry. As Kp-8 raised LH concentration, it could activate Kiss1r and stimulate the reproductive axis. As Kiss1r is associated with hyperlocomotion, it is more likely that neuropeptide FF receptor activation is involved in the suppression of locomotor activity.
ABSTRACT
Investigation of human neurodegeneration-related aggregates of beta-amyloid 1-42 (Aß42) on bdelloid rotifers is a novel interdisciplinary approach in life sciences. We reapplied an organ size-based in vivo monitoring system, exploring the autocatabolism-related alterations evoked by Aß42, in a glucose-supplemented starvation model. The experientially easy-to-follow size reduction of the bilateral reproductive organ (germovitellaria) in fasted rotifers was rescued by Aß42, serving as a nutrient source- and peptide sequence-specific attenuator of the organ shrinkage phase and enhancer of the regenerative one including egg reproduction. Recovery of the germovitellaria was significant in comparison with the greatly shrunken form. In contrast to the well-known neurotoxic Aß42 (except the bdelloids) with specific regulatory roles, the artificially designed scrambled version (random order of amino acids) was inefficient in autocatabolism attenuation, behaving as negative control. This native Aß42-related modulation of the 'functionally reversible organ shrinkage' can be a potential experiential and supramolecular marker of autocatabolism in vivo.
ABSTRACT
Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer's disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aß) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Memory/drug effects , Nerve Tissue Proteins/metabolism , Neuronal Plasticity/drug effects , Nuclear Proteins/metabolism , Oligopeptides/pharmacology , Alzheimer Disease/metabolism , Animals , Female , Male , Mice , Mice, Transgenic , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Oligopeptides/chemistry , Protein ConformationABSTRACT
Current evidence suggests dementia and pathology in Alzheimer's Disease (AD) are both dependent and independent of amyloid processing and can be induced by multiple 'hits' on vital neuronal functions. Type 2 diabetes (T2D) poses the most important risk factor for developing AD after ageing and dysfunctional IR/PI3K/Akt signalling is a major contributor in both diseases. We developed a model of T2D, coupling subdiabetogenic doses of streptozotocin (STZ) with a human junk food (HJF) diet to more closely mimic the human condition. Over 35 weeks, this induced classic signs of T2D (hyperglycemia and insulin dysfunction) and a modest, but stable deficit in spatial recognition memory, with very little long-term modification of proteins in or associated with IR/PI3K/Akt signalling in CA1 of the hippocampus. Intracerebroventricular infusion of soluble amyloid beta 42 (Aß42) to mimic the early preclinical rise in Aß alone induced a more severe, but short-lasting deficits in memory and deregulation of proteins. Infusion of Aß on the T2D phenotype exacerbated and prolonged the memory deficits over approximately 4 months, and induced more severe aberrant regulation of proteins associated with autophagy, inflammation and glucose uptake from the periphery. A mild form of environmental enrichment transiently rescued memory deficits and could reverse the regulation of some, but not all protein changes. Together, these data identify mechanisms by which T2D could create a modest dysfunctional neuronal milieu via multiple and parallel inputs that permits the development of pathological events identified in AD and memory deficits when Aß levels are transiently effective in the brain.
Subject(s)
Alzheimer Disease/epidemiology , Alzheimer Disease/etiology , Diabetes Mellitus, Type 2/complications , Alzheimer Disease/blood , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/administration & dosage , Animals , Blood Glucose/metabolism , Diabetes Mellitus, Type 2/blood , Feeding Behavior , Hippocampus/pathology , Hippocampus/ultrastructure , Humans , Insulin/blood , Male , Memory , Memory Disorders/complications , Models, Biological , Phosphorylation , Rats, Sprague-Dawley , Risk Factors , Streptozocin , Weight GainABSTRACT
Alzheimer's disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid ß (Aß) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aß oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural ß-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aß oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aß, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aß toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aß binding.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/chemistry , Dendrimers/chemistry , Peptide Fragments/chemistry , Alzheimer Disease/genetics , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Protein Precursor/antagonists & inhibitors , Amyloid beta-Protein Precursor/chemistry , Animals , Calorimetry , Dendrimers/therapeutic use , Humans , Ligands , Neurons/chemistry , Neurons/drug effects , Peptide Fragments/therapeutic use , Protein Binding , Protein Conformation/drug effects , Protein Folding/drug effectsABSTRACT
A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Oligopeptides/pharmacology , Peptide Fragments/antagonists & inhibitors , Peptidomimetics/pharmacology , Protein Conformation, alpha-Helical/drug effects , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Cell Line, Tumor , Humans , Molecular Docking Simulation , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Oligopeptides/toxicity , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptidomimetics/chemical synthesis , Peptidomimetics/metabolism , Peptidomimetics/toxicity , Protein BindingABSTRACT
Neurodegenerative diseases are linked to a systemic enzyme resistance of toxic aggregated molecules and their pathological consequences. This paper presents a unique phenomenon that Philodina acuticornis, a bdelloid rotifer, is able to catabolize different types of neurotoxic peptide and protein aggregates (such as beta-amyloids /Aß/, alpha-synuclein, and prion) without suffering any damage. P. acuticornis is capable of using these aggregates as an exclusive energy source (i.e., as 'food', identified in the digestive system and body) in a hermetically isolated microdrop environment, increasing their survival. As regards Aß1-42, five other bdelloid rotifer species were also found to be able to perform this phenomenon. Based on our experiments, the Aß1-42-treated bdelloid rotifers demonstrate significantly increased survival (e.g. mean lifespan = 51 ± 2.71 days) compared to their untreated controls (e.g. mean lifespan = 14 ± 2.29 days), with similar improvements in a variety of phenotypic characteristics. To our knowledge, no other animal species have so far been reported to have a similar capability. For all other microscopic species tested, including monogonant rotifers and non-rotifers, the treatment with Aß1-42 aggregates proved to be either toxic or simply ineffective. This paper describes and proves the existence of an unprecedented in vivo catabolic capability of neurotoxic aggregates by bdelloid rotifers, with special focus on P. acuticornis. Our results may provide the basis for a new preclinical perspective on therapeutic research in human neurodegenerative diseases.
Subject(s)
Amyloid beta-Peptides/metabolism , Rotifera/metabolism , Amyloid beta-Peptides/toxicity , Animals , Caenorhabditis elegans/metabolism , Cell Line, Tumor , Humans , Kaplan-Meier Estimate , Lobosea/metabolism , Metabolism , Neurodegenerative Diseases/metabolism , Oligohymenophorea/metabolism , Platyhelminths/metabolism , Protein Aggregation, Pathological/metabolism , Species Specificity , Tardigrada/metabolismABSTRACT
The aim of the present study was to investigate the potential anxiolytic- and antidepressant-like actions of Urocortin 2 (Ucn2) and its two fragments, Ucn2 (1-21) and Ucn2 (22-38), in mice, in an attempt to identify the biologically active sequence of this 38 amino acid neuropeptide. In this purpose, male C57BL/6 mice were treated intracerebroventricularly (icv) with 0.125, 0.25, 0.5 and 1⯵g/2⯵l of Ucn2, Ucn2 (1-21) or Ucn2 (22-38). After 30â¯min, the mice were evaluated in an elevated plus-maze test and a forced swim test for anxiety- and depression-like behavior, respectively. Each test lasted 5â¯min. Ucn2 at dose of 0.25⯵g/2⯵l and Ucn2 (1-21) at dose of 0.125⯵g/2⯵l, but not Ucn2 (22-38), increased significantly the number of entries into and the time spent in the open-arms, without influencing the total number of entries. In parallel, the same doses of Ucn2 and Ucn2 (1-21), but not Ucn2 (22-38), increased significantly the climbing and the swimming activity, while decreasing significantly the time of immobility. In addition, Ucn2 at doses of 0.125⯵g/2⯵l and 0.5⯵g/2⯵l decreased significantly the time of immobility, but they did not change the other parameters. The present study demonstrates that Ucn2 exerts anxiolytic- and antidepressant-like effects in C57BL/6 mice, which are mediated by the N-terminal, but not the C-terminal fragment of the peptide. The establishment of the smallest active sequence by further fragmentation of Ucn2 (1-21) may allow the synthesis of new anxiolytic and antidepressant drugs.
Subject(s)
Anti-Anxiety Agents/therapeutic use , Antidepressive Agents/therapeutic use , Anxiety/drug therapy , Depression/drug therapy , Urocortins/therapeutic use , Animals , Anxiety/physiopathology , Depression/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Freezing Reaction, Cataleptic/drug effects , Injections, Intraventricular , Male , Maze Learning/drug effects , Mice , Mice, Inbred C57BL , Peptides/therapeutic use , Swimming/psychology , Urocortins/chemistryABSTRACT
The function, regulation and cellular distribution of GABAA receptor subunits have been extensively documented in the adult rodent brain and are linked to numerous neurological disorders. However, there is a surprising lack of knowledge on the cellular (sub-) distribution of GABAA receptor subunits and of their expressional regulation in developing healthy and diseased foetal human brains. To propose a role for GABAA receptor subunits in neurodevelopmental disorders, we studied the developing hippocampus of normal and Down syndrome foetuses. Among the α1-3 and γ2 subunits probed, we find significantly altered expression profiles of the α1, α3 and γ2 subunits in developing Down syndrome hippocampi, with the α3 subunit being most affected. α3 subunits were selectively down-regulated in all hippocampal subfields and developmental periods tested in Down syndrome foetuses, presenting a developmental mismatch by their adult-like distribution in early foetal development. We hypothesized that increased levels of the amyloid precursor protein (APP), and particularly its neurotoxic ß-amyloid (1-42) fragment, could disrupt α3 gene expression, likely by facilitating premature neuronal differentiation. Indeed, we find increased APP content in the hippocampi of the Down foetuses. In a corresponding cellular model, soluble ß-amyloid (1-42) administered to cultured SH-SY5Y neuroblastoma cells, augmented by retinoic acid-induced differentiation towards a neuronal phenotype, displayed a reduction in α3 subunit levels. In sum, this study charts a comprehensive regional and subcellular map of key GABAA receptor subunits in identified neuronal populations in the hippocampus of healthy and Down syndrome foetuses and associates increased ß-amyloid load with discordant down-regulation of α3 subunits.
Subject(s)
Down Syndrome/pathology , Gene Expression Regulation, Developmental/physiology , Hippocampus/metabolism , Hippocampus/pathology , Protein Subunits/metabolism , Receptors, GABA-A/metabolism , Adolescent , Adult , Age Factors , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/pharmacology , Animals , Animals, Newborn , Case-Control Studies , Doublecortin Domain Proteins , Down Syndrome/genetics , Embryo, Mammalian , Female , Fetus , Gene Expression Regulation, Developmental/drug effects , Gestational Age , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/metabolism , Hippocampus/growth & development , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Middle Aged , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuroblastoma/pathology , Neurons/drug effects , Neurons/metabolism , Neuropeptides/metabolism , POU Domain Factors/genetics , POU Domain Factors/metabolism , Peptide Fragments/pharmacology , Protein Subunits/genetics , Tretinoin/pharmacology , Vesicular Inhibitory Amino Acid Transport Proteins/metabolism , Young AdultABSTRACT
During the past 15 years, several genetically altered mouse models of human Alzheimer's disease (AD) have been developed. These costly models have greatly facilitated the evaluation of novel therapeutic approaches. Injecting synthetic ß-amyloid (Aß) 1-42 species into different parts of the brain of non-transgenic rodents frequently provided unreliable results, owing to a lack of a genuine characterization of the administered Aß aggregates. Previously, we have published a new rat AD-model in which protofibrillar-fibrillar Aß1-42 was administered into rat entorhinal cortex (Sipos 2007). In order to develop a more reliable model, we have injected well-characterized toxic soluble Aß1-42 species (oligomers, protofibrils and fibrils) intracerebroventricularly (icv) into rat brain. Studies of the distribution of fluorescent-labeled Aß1-42 in the brain showed that soluble Aß-species diffused into all parts of the rat brain. After seven days, the Aß-treated animals showed a significant decrease of spatial memory in Morris water maze test and impairment of synaptic plasticity (LTP) measured in acute hippocampal slices. The results of histological studies (decreased number of viable neurons, increased tau levels and decreased number of dendritic spines) also supported that icv administration of well-characterized toxic soluble Aß species into rat brain provides a reliable rat AD-model.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/therapeutic use , Spatial Memory/drug effects , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/administration & dosage , Animals , Disease Models, Animal , Male , Maze Learning/drug effects , RatsABSTRACT
BACKGROUND: The blood-brain barrier restricts drug penetration to the central nervous system. Targeted nanocarriers are new potential tools to increase the brain entry of drugs. Ligands of endogenous transporters of the blood-brain barrier can be used as targeting vectors for brain delivery of nanoparticles. OBJECTIVE: We tested biotin-labeled solid nanoparticles for the first time and compared to biotinylated glutathione- labeled nanoparticles in brain endothelial cells. METHOD: Neutravidin coated fluorescent polystyrene nanoparticles were derivatized with biotin and biotinylated glutathione. As a human in vitro blood-brain barrier model hCMEC/D3 brain endothelial cells were used. Cell viability by MTT test, uptake and transfer of the nanoparticles across the endothelial monolayers were measured. The uptake of the nanoparticles was visualized by confocal microscopy. RESULTS: The tested nanoparticles caused no change in cell viability. The uptake of biotin- and glutathione-labeled nanoparticles by brain endothelial cells was time-dependent and significantly higher compared to non-labeled nanoparticles. The penetration of the glutathione-labeled nanoparticles across the endothelial monolayer was higher than the biotin-targeted ones. Biotin- and glutathione-targeted nanoparticles were visualized in hCMEC/D3 cells. We verified that hCMEC/D3 express mRNA for sodium-dependent multivitamin transporter (SMVT/SLC5A6) responsible for the blood-brain barrier transport of biotin. CONCLUSION: Biotin as a ligand increased the uptake and the transfer of nanoparticles across brain endothelial cells. Biotinylated glutathione could further increase nanoparticle permeability through endothelial monolayers supporting its use as a brain targeting vector.
Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , Drug Delivery Systems , Animals , Biological Transport , Biotin/administration & dosage , Cell Survival , Endothelial Cells/metabolism , Glutathione/administration & dosage , Humans , Nanoparticles , Tissue DistributionABSTRACT
Accumulation of amyloid plaques comprises one of the major hallmarks of Alzheimer's disease (AD). In rodents, acute treatment with amyloid-beta (Aß; 1-42) elicits immediate debilitating effects on hippocampal long-term potentiation (LTP). Whereas LTP contributes to synaptic information storage, information is transferred across neurons by means of neuronal oscillations. Furthermore, changes in theta-gamma oscillations, that appear during high-frequency stimulation (HFS) to induce LTP, predict whether successful LTP will occur. Here, we explored if intra-cerebral treatment with Aß(1-42), that prevents LTP, also results in alterations of hippocampal oscillations that occur during HFS of the perforant path-dentate gyrus synapse in 6-month-old behaving rats. HFS resulted in LTP that lasted for over 24 h. In Aß-treated animals, LTP was significantly prevented. During HFS, spectral power for oscillations below 100 Hz (δ, θ, α, ß and γ) was significantly higher in Aß-treated animals compared to controls. In addition, the trough-to-peak amplitudes of theta and gamma cycles were higher during HFS in Aß-treated animals. We also observed a lower amount of envelope-to-signal correlations during HFS in Aß-treated animals. Overall, the characteristic profile of theta-gamma oscillations that accompany successful LTP induction was disrupted. These data indicate that alterations in network oscillations accompany Aß-effects on hippocampal LTP. This may comprise an underlying mechanism through which disturbances in synaptic information storage and hippocampus-dependent memory occurs in AD.
ABSTRACT
Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disorders characterized by neuritic plaques containing amyloid-ß peptide (Aß) and neurofibrillary tangles. Evidence has been reported that Aß(1-42) plays an essential pathogenic role in decreased spine density, impairment of synaptic plasticity, and neuronal loss with disruption of memory-related synapse function, all associated with AD. Experimentally, Aß(1-42) oligomers perturb hippocampal long-term potentiation (LTP), an electrophysiological correlate of learning and memory. Aß was also reported to perturb synaptic glutamate (Glu)-recycling by inhibiting excitatory-amino-acid-transporters. Elevated level of extracellular Glu leads to activation of perisynaptic receptors, including NR2B subunit containing NMDARs. These receptors were shown to induce impaired LTP and enhanced long-term depression and proapoptotic pathways, all central features of AD. In the present study, we investigated the role of Glu-recycling on Aß(1-42)-induced LTP deficit in the CA1. We found that Aß-induced LTP damage, which was mimicked by the Glu-reuptake inhibitor TBOA, could be rescued by blocking the NR2B subunit of NMDA receptors. Furthermore, decreasing the level of extracellular Glu using a Glu scavenger also restores TBOA or Aß induces LTP damage. Overall, these results suggest that reducing ambient Glu in the brain can be protective against Aß-induced synaptic disruption.
Subject(s)
Amyloid beta-Peptides/pharmacology , Glutamic Acid/metabolism , Neuronal Plasticity/drug effects , Peptide Fragments/pharmacology , Synapses/drug effects , Alanine Transaminase/pharmacology , Analysis of Variance , Animals , Aspartic Acid/pharmacology , Biophysics , Electric Stimulation , Excitatory Amino Acid Antagonists/pharmacology , Hippocampus/cytology , In Vitro Techniques , Mice , Mice, Inbred BALB C , Nerve Net/drug effects , Patch-Clamp Techniques , Piperidines/pharmacology , Pyruvic Acid/pharmacologyABSTRACT
Neuronal hyperexcitability is a phenomenon associated with early Alzheimer's disease. The underlying mechanism is considered to involve excessive activation of glutamate receptors; however, the exact molecular pathway remains to be determined. Extracellular recording from the CA1 of hippocampal slices is a long-standing standard for a range of studies both in basic research and in neuropharmacology. Evoked field potentials (fEPSPs) are regarded as the input, while spiking rate is regarded as the output of the neuronal network; however, the relationship between these two phenomena is not fully clear. We investigated the relationship between spontaneous spiking and evoked fEPSPs using mouse hippocampal slices. Blocking AMPA receptors (AMPARs) with CNQX abolished fEPSPs, but left firing rate unchanged. NMDA receptor (NMDAR) blockade with MK801 decreased neuronal spiking dose dependently without altering fEPSPs. Activating NMDARs by small concentration of NMDA induced a trend of increased firing. These results suggest that fEPSPs are mediated by synaptic activation of AMPARs, while spontaneous firing is regulated by the activation of extrasynaptic NMDARs. Synaptotoxic Abeta(1-42) increased firing activity without modifying evoked fEPSPs. This hyperexcitation was prevented by ifenprodil, an antagonist of the NR2B NMDARs. Overall, these results suggest that Abeta(1-42) induced neuronal overactivity is not dependent on AMPARs but requires NR2B.