66 research outputs found

    Discovering the signalling cues involved in early human brain development

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    Enhancing memory-related sleep spindles through learning and electrical brain stimulation

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    Sleep has been strongly implicated in mediating memory consolidation through hippocampal-neocortical communication. Evidence suggests offline processing of encoded information in the brain during slow wave sleep (SWS), specifically during slow oscillations and spindles. In this work, we used active exploration and learning tasks to study post-experience sleep spindle density changes in rats. Experiences lead to subsequent changes in sleep spindles, but the strength and timing of the effect was task-dependent. Brain stimulation in humans and rats have been shown to enhance memory consolidation. However, the exact stimulation parameters which lead to the strongest memory enhancement have not been fully explored. We tested the efficacy of both cortical sinusoidal direct current stimulation and intracortical pulse stimulation to enhance slow oscillations and spindle density. Pulse stimulation reliably evoked state-dependent slow oscillations and spindles during SWS with increased hippocampal ripple-spindle coupling, demonstrating potential in memory enhancement

    Discovery and characterisation of anti-amyloid beta fibril antibodies from naive phage display VHH libraries

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    Parkinson’s disease (PD) and Alzheimer’s disease (AD) are two of the most common incurable neurological disorders affecting the worldwide population over 60 years of age and are characterized by the progressive loss of either motor or cognitive functions in affected individuals. Although the main cause of these diseases remains unknown, genetic, histological and animal models point to the progressive accumulation of misfolded synaptic proteins called alpha-synuclein (α-syn) and amyloid beta (Aβ) as the main suspects in, respectively, causing PD or AD. In pathological conditions, α-syn and Aβ have been observed to change their physiological conformations and aggregate into protein polymers, also known as fibrils. These different structures have been directly linked to disease progression, and thus the in depth understanding of these polymers is crucial for future hopes of identifying a cure for PD or AD. Currently, no drugs have been identified with the ability to reverse or reduce the disease burden of these diseases, with available therapies only delaying the inevitable progression of the diseases. During this project, a way to reproduce these disease-relevant structures was identified through the means of protein misfolding cyclic amplification (PMCA) and real-time quaking induced conversion (RT-QUIC), two methodologies developed for the amplification of protein polymers in vitro. By means of different biochemical and imaging methods two distinct α-syn polymorphs and two Aβ conformers were detected. Next, a naïve VHH library was implemented to discover antibodies against the characterized Aβ fibrils through several rounds of biopanning. This was done for both full-length fibril conformers (or polymorphs) and fragmented fibrils, with the latter strategy targeting the elongation sites responsible for fibril propagation. Overall, an array of antibodies were discovered that bound to fibrils or fragmented fibrils. The binding properties of these antibodies were then characterized through immunoassays and the measuring of biomolecular interactions with bio-layer interferometry. This was achieved using both fibril polymorphs, fragmented fibrils, monomers (sourced from one supplier, Genscript) and a mixed solution of monomers, oligomers and protofibrils sourced from another supplier (Gencust). From this analysis it was revealed that fibril binders could be grouped in three categories, depending on their binding affinity to each of the different Aβ forms tested: 1) binders to all forms tested (including fibrils, monomers, oligomers and protofibrils); 2) binders to both fibril polymorphs and the mixed aggregate solution from Gencust and 3) binders to a single fibril polymorph and Gencust monomers, oligomers and protofibrils. Fragmented fibril binders, on the other hand, could be grouped in 4 categories: 1) binders to fragmented fibrils, both fibril conformers and Gencust monomers, oligomers and protofibrils; 2) binders to fragmented fibrils, one of the fibril polymorphs and Gencust monomers, oligomers and protofibrils; 3) binders to a single fibril polymorph and Gencust monomers, oligomers and protofibrils and 4) binders to all forms of Aβ tested (including fragmented and both full-length fibril polymorphs, Genscript monomers and Gencust monomers, oligomers and protofibrils). Functional assays were then attempted for 9 antibodies, producing preliminary data demonstrating that the VHH antibodies identified through phage display might have a protective effect in vitro with the inhibition of the formation of fibrils in solution

    Models and Analysis of Vocal Emissions for Biomedical Applications

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    The International Workshop on Models and Analysis of Vocal Emissions for Biomedical Applications (MAVEBA) came into being in 1999 from the particularly felt need of sharing know-how, objectives and results between areas that until then seemed quite distinct such as bioengineering, medicine and singing. MAVEBA deals with all aspects concerning the study of the human voice with applications ranging from the neonate to the adult and elderly. Over the years the initial issues have grown and spread also in other aspects of research such as occupational voice disorders, neurology, rehabilitation, image and video analysis. MAVEBA takes place every two years always in Firenze, Italy. This edition celebrates twenty years of uninterrupted and succesfully research in the field of voice analysis

    Exploring the Role of PANNEXIN1A in an Acute Experimental Model of Parkinson's Disease

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    In the nervous system Pannexin1 channels are major ATP and glutamate release sites. The channels are implicated in neurodegenerative disorders including Parkinson’s disease, but the underlying mechanisms remain unclear. Here, an interdisciplinary approach tested roles of the mammalian Pannexin1 ortholog Pannexin1a in a zebrafish model of MPTP-induced early stages of Parkinson’s disease at molecular, systems, and behavioral levels. The short-term treatment of wild-type TL and gene-edited Panx1a-KO larvae caused metabolic stress, regulated inflammatory pathways, and reduced ATP production. Local field potentials recorded from three regions of the ascending visual pathway showed complex changes in the beta- and gamma-band power and in the coherence between these regions. MPTP treatment produced significantly impaired movements which were partially rescued by targeting the NLRP3 inflammasome. The main findings of this research provide evidence that Panx1a serves a neuroprotective role in an acute MPTP model of Parkinson's disease

    Exploring the Role of PANNEXIN1A in an Acute Experimental Model of Parkinson's Disease

    Get PDF
    In the nervous system Pannexin1 channels are major ATP and glutamate release sites. The channels are implicated in neurodegenerative disorders including Parkinsons disease, but the underlying mechanisms remain unclear. Here, an interdisciplinary approach tested roles of the mammalian Pannexin1 ortholog Pannexin1a in a zebrafish model of MPTP-induced early stages of Parkinsons disease at molecular, systems, and behavioral levels. The short-term treatment of wild-type TL and gene-edited Panx1a-KO larvae caused metabolic stress, regulated inflammatory pathways, and reduced ATP production. Local field potentials recorded from three regions of the ascending visual pathway showed complex changes in the beta- and gamma-band power and in the coherence between these regions. MPTP treatment produced significantly impaired movements which were partially rescued by targeting the NLRP3 inflammasome. The main findings of this research provide evidence that Panx1a serves a neuroprotective role in an acute MPTP model of Parkinson's disease

    Impact of LRRK2 kinase activation and inhibition in vivo and in vitro

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    Parkinson’s Disease (PD) is a progressive neurodegenerative disorder that is characterized in part by a loss of dopamine neurons in the Substantia Nigra and affects the nigrostriatal pathway. Mutations in the gene encoding leucine�rich repeat kinase 2 (LRRK2) have been found to cause late onset PD through a gain of function of its kinase domain. Thus, LRRK2 has become an intriguing candidate for therapeutic intervention by kinase inhibition. While preclinical studies have shown that ablating the kinase activity of wildtype LRRK2 is safe with a mild and reversible lung phenotype, the molecular effects of chronic LRRK2 inhibition have not been examined in the context of mutant LRRK2. Using the potent LRRK2 kinase-specific inhibitor, Merck LRRK2 inhibitor 2 (MLi�2), hyperactive G2019S LRRK2 was reduced to wildtype levels chronically in G2019S knock-in (KI) mice and autophosphorylation of LRRK2 and phosphorylation of direct substrates Rab10, Rab12, and Rab29 was assessed. Unbiased total and phospho-proteomics revealed alterations in endolysosomal proteins similar to those found in LRRK2 knockout (KO) animals after 10 weeks of treatment. LRRK2 has been shown to play roles in a number of pathways within the endolysosomal system and studies have reported LRRK2 presence on many organelles from the trans-Golgi network (TGN) to the lysosome. Based on the current study’s proteomic results, eight different trap plasmids were generated to evaluate LRRK2 kinase activity at distinct endolysosomal membranes. As a result, LRRK2 kinase activity was found to be enhanced after being trapped to all membranes and downstream Rab10 and Rab12 phosphorylation were increased in vitro, but recruitment of these Rabs revealed differential patterns in localization specifically when targeting LRRK2 on lysosomes compared to other membranes. Evaluation of lysosomal position through manipulation of various motor proteins showed phosphorylated Rab10 was preferentially restricted to a subset of perinuclear lysosomes, whereas pRab12 was present at most LRRK2-positive lysosomes regardless of their position. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular pathways that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients. Complementary in vitro data provides novel insight into the differences in LRRK2-dependent Rab localization that can help elucidate the role of LRRK2 at the lysosome which may be relevant to PD pathogenesis. The work presented in this thesis additionally contributes to our knowledge on the utility of pS1292 LRRK2 and pS106 Rab12 as robust biomarkers of both kinase hyperactivity and inhibition in G2019S LRRK2 KI mice in brain and peripheral tissues that is worth assessment in patients with PD harboring the G2019S mutation
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