4,229 research outputs found
When, where and how? Focus on neuronal calcium dysfunctions in Alzheimer's Disease.
Alzheimer\u2019s disease (AD), since its characterization as a precise form of dementia with its own pathological
hallmarks, has captured scientists\u2019 attention because of its complexity. The last 30 years have been
filled with discoveries regarding the elusive aetiology of this disease and, thanks to advances in molecular
biology and live imaging techniques, we now know that an important role is played by calcium (Ca2+).
Ca2+, as ubiquitous second messenger, regulates a vast variety of cellular processes, from neuronal excitation
and communication, to muscle fibre contraction and hormone secretion, with its action spanning
a temporal scale that goes from microseconds to hours. It is therefore very challenging to conceive a
single hypothesis that can integrate the numerous findings on this issue with those coming from the
classical fields of AD research such as amyloid-beta (A) and tau pathology. In this contribution, we will
focus our attention on the Ca2+ hypothesis of AD, dissecting it, as much as possible, in its subcellular
localization, where the Ca2+ signal meets its specificity. We will also follow the temporal evolution of the
Ca2+ hypothesis, providing some of the most updated discoveries. Whenever possible, we will link the
findings regarding Ca2+ dysfunction to the other players involved in AD pathogenesis, hoping to provide
a crossover body of evidence, useful to amplify the knowledge that will lead towards the discovery of an
effective therapy
Phytocannabinoids as novel therapeutic agents in CNS disorders
The Cannabis sativa herb contains over 100 phytocannabinoid (pCB) compounds and has been used for thousands of years for both recreational and medicinal purposes. In the past two decades, characterisation of the body's endogenous cannabinoid (CB) (endocannabinoid, eCB) system (ECS) has highlighted activation of central CB1 receptors by the major pCB, Δ9-tetrahydrocannabinol (Δ9-THC) as the primary mediator of the psychoactive, hyperphagic and some of the potentially therapeutic properties of ingested cannabis. Whilst Δ9-THC is the most prevalent and widely studied pCB, it is also the predominant psychotropic component of cannabis, a property that likely limits its widespread therapeutic use as an isolated agent. In this regard,
research focus has recently widened to include other pCBs including cannabidiol (CBD), cannabigerol (CBG), Δ9tetrahydrocannabivarin (Δ9-THCV) and cannabidivarin (CBDV), some of which show potential as therapeutic agents in preclinical models of CNS disease. Moreover, it is becoming evident that these non-Δ9-THC pCBs act at a wide range of pharmacological targets, not solely limited to CB receptors. Disorders that could be targeted include epilepsy, neurodegenerative diseases, affective disorders and the central modulation of feeding behaviour. Here, we review pCB effects in preclinical models of CNS disease and, where available, clinical trial data that support therapeutic effects. Such developments may soon yield the first non-Δ9-THC pCB-based medicines
A Knowledge-based Integrative Modeling Approach for <em>In-Silico</em> Identification of Mechanistic Targets in Neurodegeneration with Focus on Alzheimer’s Disease
Dementia is the progressive decline in cognitive function due to damage or disease in the body beyond what might be expected from normal aging. Based on neuropathological and clinical criteria, dementia includes a spectrum of diseases, namely Alzheimer's dementia, Parkinson's dementia, Lewy Body disease, Alzheimer's dementia with Parkinson's, Pick's disease, Semantic dementia, and large and small vessel disease. It is thought that these disorders result from a combination of genetic and environmental risk factors. Despite accumulating knowledge that has been gained about pathophysiological and clinical characteristics of the disease, no coherent and integrative picture of molecular mechanisms underlying neurodegeneration in Alzheimer’s disease is available. Existing drugs only offer symptomatic relief to the patients and lack any efficient disease-modifying effects. The present research proposes a knowledge-based rationale towards integrative modeling of disease mechanism for identifying potential candidate targets and biomarkers in Alzheimer’s disease. Integrative disease modeling is an emerging knowledge-based paradigm in translational research that exploits the power of computational methods to collect, store, integrate, model and interpret accumulated disease information across different biological scales from molecules to phenotypes. It prepares the ground for transitioning from ‘descriptive’ to “mechanistic” representation of disease processes. The proposed approach was used to introduce an integrative framework, which integrates, on one hand, extracted knowledge from the literature using semantically supported text-mining technologies and, on the other hand, primary experimental data such as gene/protein expression or imaging readouts. The aim of such a hybrid integrative modeling approach was not only to provide a consolidated systems view on the disease mechanism as a whole but also to increase specificity and sensitivity of the mechanistic model by providing disease-specific context. This approach was successfully used for correlating clinical manifestations of the disease to their corresponding molecular events and led to the identification and modeling of three important mechanistic components underlying Alzheimer’s dementia, namely the CNS, the immune system and the endocrine components. These models were validated using a novel in-silico validation method, namely biomarker-guided pathway analysis and a pathway-based target identification approach was introduced, which resulted in the identification of the MAPK signaling pathway as a potential candidate target at the crossroad of the triad components underlying disease mechanism in Alzheimer’s dementia
Hybrid Adult Neuron Culture Systems for Use in Pharmacological Testing
Neuronal culture systems have many applications, such as basic research into neuronal structure, function, and connectivity as well as research into diseases, conditions, and injuries affecting the brain and its components. In vitro dissociated neuronal systems have typically been derived from embryonic brain tissue, most commonly from the hippocampus of E18 rats. This practice has been motivated by difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. The overall focus of this dissertation research was to develop a dissociated neuronal culture system from human and animal adult brain tissue, one more functionally and developmentally correlative to the mature brain. To that end, this work was divided into five interrelated topics: development of an adult in vitro neuronal culture system comprised of electrically functional, mitotically stable, developmentally mature neurons from the hippocampus of adult rats; creation of stable two-cell neuronal networks for the study of synaptic communication in vitro; coupling of electrically active adult neurons to microelectrode arrays for high-throughput data collection and analysis; identification of inadequacies in embryonic neuronal culture systems and proving that adult neuronal culture systems were not deficient in similar areas; augmentation of the rat hippocampal culture system to allow for the culture and maintenance of electrically active human neurons for months in vitro. The overall hypothesis for this dissertation project was that tissue engineered in vitro systems comprised of neurons dissociated from mature adult brain tissue could be developed using microfabrication, defined medium formulations, optimized culture and maintenance parameters, and cell-cycle control. Mature differentiated glutamatergic neurons were extracted from hippocampal brain tissue and processed to purify neurons and remove tissue debris. Terminally differentiated rat hippocampal neurons recovered in vitro and displayed mature neuronal morphology. Extracellular glutamate in the culture medium promoted neuronal recovery of electrical function and activity. After recovery, essential growth factors in the culture medium caused adult neurons to reenter the cell cycle and divide multiple times. Only after reaching confluence did some neurons stop dividing. Strategies for inhibition of neuronal mitotic division were investigated, and manipulation of the cdk5 pathway was ultimately found to prevent division in vitro. Prevention of mitotic division as well as optimization of culture and maintenance parameters resulted in a neuronal culture system derived from adult rats in which the neuronal morphology, cytoskeleton and surface protein expression patterns, and electrical activity closely mirrored mature, terminally differentiated adult neurons in vivo. Improvements were also made to the growth surfaces on which neurons attached, regenerated, and survived long-term. Culture surfaces, in this case glass cover slips, were modified with the chemical substrate N-1 (3-(trimethoxysilyl) propyl)-diethylenetriamine (DETA) to create a covalently modified interface with exposed cell-adhesive triamine groups. DETA chemical surfaces were also further modified to create high-resolution patterns, useful in creating engineered two-cell networks of adult hippocampal neurons. Adult hippocampal neurons were also coupled to microelectrode array systems (MEAs) and recovered functionally, fired spontaneously, and reacted to synaptic antagonists in a manner consistent to adult neurons in vivo. Last, neurons from the brains of deceased Alzheimer\u27s disease (AD) patients and from brain tissue excised during surgery for Parkinson\u27s disease (PD), Essential Tremor (ET), and brain tumor were isolated and cultured, with these neurons morphological regenerating and electrically recovering in vitro
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Automated parallel immobilization microfluidic platforms for high-throughput neuronal degeneration studies with C. elegans
C. elegans has emerged as an invaluable model organism for in vivo neurobiology research to understand disease mechanisms and pathology relevant in humans. Simple anatomy, short lifecycle, fully characterized genome, and miniature body scale make these nematodes an ideal model organism for phenotyping and bio-molecular studies using microfluidic platforms. Advancements in soft-lithography have improved the functionality of microfluidic technology for C. elegans, leading to whole organism studies in high-throughput manner that were not otherwise possible. In order to study phenomena that require large amounts of data such as drug screens for neurological disorders and phenotyping, high-throughput imaging platforms with high-speed, high-resolution image acquisition become essential. With this in mind, we have developed and tested microfluidic immobilization devices to enable high-throughput optical interrogation of C. elegans for neurodegenerative diseases and large scale drug screens. Initially, we designed, developed, and tested single-layer and double-layer SU8 mold PDMS chips with parallel tapered channels to immobilize 40 adult C. elegans for high-resolution fluorescence imaging of their neurons in a parallel manner. vi We achieved over 90% immobilization efficiency using these initial devices, but could achieve only ~50% of the trapped worms with proper orientation to allow scoring of the VC neurons of interest. To improve worm orientation, we developed a three-layer microfluidic chip that can immobilize and orient the adult worms for optical interrogation of these VC neurons with 90% efficiency. Finally, we scaled the platform to accommodate a large scale platform with standard multi-well format on-chip wells where each well leads to the optimized trapping channels. The final optimized multi-well platform provides comprehensive easy to use 96-well microfluidic system to orient, immobilize, and image adult C. elegans in high-throughput manner. The novel gasket system can pressurize the multi-well device pre-loaded with 96 individual worm populations. Using a sequence of on-off applied gasket pressure, we can orient and immobilize worms in all 96 devices simultaneously in less than 5 minutes. Custom designed software can capture 12 z-stack images per worm from all 96-well in less than 12 minutes. With 95% trapping efficiency, approximately 90% of the worms can be scored successfully for neuronal phenotyping of VC neurons. This 96-well platform and the automated imaging system enable high-throughput optical interrogation of adult C. elegans for large-scale drug screens relating to ageing and various neurodegenerative diseases.Mechanical Engineerin
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Ischemic axonal injury up-regulates MARK4 in cortical neurons and primes tau phosphorylation and aggregation.
Ischemic injury to white matter tracts is increasingly recognized to play a key role in age-related cognitive decline, vascular dementia, and Alzheimer's disease. Knowledge of the effects of ischemic axonal injury on cortical neurons is limited yet critical to identifying molecular pathways that link neurodegeneration and ischemia. Using a mouse model of subcortical white matter ischemic injury coupled with retrograde neuronal tracing, we employed magnetic affinity cell sorting with fluorescence-activated cell sorting to capture layer-specific cortical neurons and performed RNA-sequencing. With this approach, we identified a role for microtubule reorganization within stroke-injured neurons acting through the regulation of tau. We find that subcortical stroke-injured Layer 5 cortical neurons up-regulate the microtubule affinity-regulating kinase, Mark4, in response to axonal injury. Stroke-induced up-regulation of Mark4 is associated with selective remodeling of the apical dendrite after stroke and the phosphorylation of tau in vivo. In a cell-based tau biosensor assay, Mark4 promotes the aggregation of human tau in vitro. Increased expression of Mark4 after ischemic axonal injury in deep layer cortical neurons provides new evidence for synergism between axonal and neurodegenerative pathologies by priming of tau phosphorylation and aggregation
Hippocampus
The hippocampus is a bicortical structure with extensive fiber connections with multiple brain regions. It is involved in several functions, such as learning, memory, attention, emotion, and more. This book covers various aspects of the hippocampus including cytoarchitecture, functions, diseases, and treatment. It highlights the most advanced findings in research on the hippocampus. It discusses circuits, pattern formation process of grid cells, and zinc dynamics of the hippocampus. The book also addresses the tau pathology and circRNAs related to Alzheimer’s disease and potential treatment strategies. It is a useful resource for general readers, students, and researchers
Etude expérimentale des dynamiques temporelles du comportement normal et pathologique chez le rat et la souris
155 p.Modern neuroscience highlights the need for designing sophisticated behavioral readout of internal cognitive states. From a thorough analysis of classical behavioral test, my results supports the hypothesis that sensory ypersensitivity might be the cause of other behavioural deficits, and confirm the potassium channel BKCa as a potentially relevant molecular target for the development of drug medication against Fragile X Syndrome/Autism Spectrum Disorders. I have also used an innovative device, based on pressure sensors that can non-invasively detect the slightest animal movement with unprecedented sensitivity and time resolution, during spontaneous behaviour. Analysing this signal with sophisticated computational tools, I could demonstrate the outstanding potential of this methodology for behavioural phenotyping in general, and more specifically for the investigation of pain, fear or locomotion in normal mice and models of neurodevelopmental and neurodegenerative disorders
Systematic comparison of the molecular mechanisms underlying the spreading of pathology in different neurodegenerative diseases
Misfolding and accumulation of disease-related proteins are common hallmarks among several neurodegenerative diseases. This phenomenon causes the progressive loss of cognitive and motor functions correlated with specific cell loss in the brain. Despite the different clinical manifestations, these disorders share common features and molecular mechanisms, such as aggregation and accumulation of disease-related proteins in specific regions of the brain. These include alpha-synuclein aggregates in Parkinson’s disease and other synucleinopathies, inclusions of hyperphosphorylated microtubule-binding Tau in tauopathies, and extended polyglutamine protein aggregates (huntingtin) in Huntington’s disease.
The association between the progression of clinical symptoms and the topographical distribution of pathology in neurodegenerative diseases has led to the hypothesis that specific pathological disease-related proteins can be transferred intercellularly in a prion-like manner. This hypothesis was created after the observation Lewy body pathology, a characteristic hallmark in synucleinopathies, within fetal dopaminergic neurons grafts in Parkinson’s disease patients. Later, injection of Tau aggregates into animal models was shown to induce pathology. More recently, similar mechanisms were proposed to occur in monogenic forms of neurodegenerative diseases after the observation of mutant huntingtin aggregates within fetal striatal allografts in the brain of Huntington’s disease patients. Furthermore, the presence of co-pathology in the brain of patients with distinct neurodegenerative diseases has implied that several proteins may overlap to contribute to the neuropathophysiology and can share common molecular mechanisms in neurodegeneration.
Several mechanisms have been suggested to contribute to the spreading of alpha-synuclein, Tau and huntingtin pathology. These include diffusion through the plasma membrane, release via extracellular vesicles (as ectosomes and exosomes), misfolded-associated protein secretion pathway, membrane carriers, membrane pores, tunnelling nanotubes, endocytosis, and receptor-mediated endocytosis. To date, it is unclear if alpha-synuclein, Tau and huntingtin release to the extracellular milieu occurs through similar cellular mechanisms and their effect in receptor cells. Also, the role of protein secretion and their involvement in neuronal dysfunction and disease progression remains elusive. Further elucidation of these questions will permit a better understanding of protein propagation on disease pathogenesis in neurodegenerative diseases.
In recent years, extracellular vesicles have emerged as central mediators in intercellular communication under physiological and pathological conditions. Their heterogeneity and presence in several human biofluids have led to extensive research regarding their content and functional properties as relevant biomarkers in neurodegenerative diseases. In particular, ectosomes and exosomes are considered important carriers of misfolded proteins between cells in disease. These can be internalized in diverse cell types, although their effect in neuronal activity is unclear. Discernment between ectosomes and exosomes has been difficult due to the moderate differences in their physical properties and absence of reliable markers. While exosomes have been extensively studied in the field, the role of ectosomes in the pathogenesis of neurodegenerative diseases has not been explored. Additional studies focusing in the role of ectosomes and other types of extracellular vesicles in neurodegenerative diseases will open new avenues to uncover potential disease biomarkers and therapeutic targets.
In our first study, we developed a detailed differential ultracentrifugation protocol to efficiently purify ectosomes and exosomes, and provided a proteomic and functional characterization of these vesicles subtypes. Comprehensive proteomic analysis revealed specific protein composition and pathways enrichment for ectosomes and exosomes. Interestingly, ectosomes isolated from human cerebrospinal fluid and from cell media displayed enrichment in annexin-A2, suggesting this protein as an important marker for ectosomes characterization. Furthermore, treatment of neuronal cortical cultures with ectosomes and exosomes resulted in their internalization at similar ratios. Using multi-electrode array technology, we further demonstrated that extracellular vesicles internalization affects differently the spontaneous activity of neuronal cells.
In our second study, we demonstrated that common cellular mechanisms are used for the transfer of alpha-synuclein, Tau and huntingtin exon-1 fragments between cells. Interestingly, we observed that these proteins are handled in different ways depending on the receptor cells. Our results reveal the release of the different disease-related proteins to the cell media at different levels in a free form and in extracellular vesicles. Overall, alpha-synuclein, Tau and normal huntingtin exon-1 were found in higher levels in the cell media in contrast to mutant huntingtin exon-1. We further observed discernible alterations in the spontaneous firing activity in primary cortical neurons after treatment with the different recombinant proteins, suggesting that the effects of alpha-synuclein, Tau and huntingtin in the extracellular space and on neuronal activity are dependent of the protein properties and not only correlated with their secretion levels. Interestingly, alpha-synuclein, Tau and huntingtin exon-1 were present in higher levels in ectosomes than in exosomes.
We revealed that these vesicles could be internalized in microglial and astrocytic cells, and resulted in the production of pro-inflammatory cytokines and autophagy activation. Neuronal cells also internalized ectosomes and exosomes enriched with alpha-synuclein, Tau or huntingtin, and exhibited irregularity in the cell bursting properties that overall was correlated with the vesicles subtype.
Overall, our work indicates that extracellular vesicles cargoes likely reflect core pathophysiological intracellular processes in their origin cells. A clear understanding of the specific functional properties of different vesicles subtypes might represent a step forward in the search of novel biomarkers. Furthermore, our results propose that common cellular mechanisms are used for the transfer of alpha-synuclein, Tau and huntingtin between cells. These similarities could suggest common therapeutic targets for neurodegenerative diseases, and the need to target several cellular mechanisms to halt the detrimental effects of protein transmission and pathology progression.2021-10-2
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