Precision mapping of gene expression and proteins in the brain using gene editing and barcoded viral vectors

The human brain is a masterpiece of intricate design and impeccable functionality. It serves as the ultimate command center for our thoughts, sensations, and actions, which define our very existence. This organ operates flawlessly, with billions of neurons working in perfect harmony to process information, create memories, and regulate our emotions. The brain's neural network is composed of trillions of connections, consisting of interconnected cells that communicate through electrical impulses and chemical signals at remarkable speeds. These connections, also known as synapses, serve as the means of communication that allow for information to travel uninterrupted throughout the brain. This intricate network enables us to think, learn, reason, and react to our surroundings. However, neurological disorders have the potential to disrupt this delicate balance, leading to a range of manifestations. These can include gradual memory erosion in Alzheimer's disease to the slow progression of motor and cognitive impairment in Parkinson's disease. Each condition presents a unique puzzle for scientists and researchers to decipher. The intricate interactions of genes, proteins, and neural circuits create a complex landscape that holds the key to understanding these disorders' origins and potential treatments.In this thesis, we worked on understanding a new type of neuronal communication based on the retrotransposon protein of Arc. The investigation was conducted using a gene editing technique based on the CRISPR/Cas9 system, next-generation sequencing technologies, and refined immunohistochemistry protocol. Using a mouse animal model, our findings reinforced the hypothesis that Arc has the capacity for inter-neuronal transport, as previously proposed in vitro studies. An additional objective of the thesis has been the investigation of molecular changes occurring within the Substantia Nigra throughout the progression of Parkinson's disease. At the core of this disorder's pathophysiology lies the alpha-synuclein protein. With this objective in mind, we developed a single- cell methodology to effectively investigate modifications in gene expression provoked by an overload of alpha- synuclein in animal models of rodents. From this data set, the overarching goal is to train a machine learning able to predict the disease course and to establish possible therapeutic interventions

La Rete Integrata Nazionale GPS (RING) dell' INGV: una infrastruttura aperta per la ricerca scientifica

Since 2004, the Istituto Nazionale di Geofisica e Vulcanologia (INGV) is investing important energies for the creation of a continuous GPS network dislocated all over the Italian territory. Data transmission will occur in real time, integrating the experiences already existing in the different INGV institutes and developing a 3-yrs strategy for the new installations. The main targets of the network are represented by active tectonics studies, including also the seismological part as strain accumulation on faults. Within a 3-yrs funding project, it is expected, to realize for the scientific community an infrastructure which is comparable to those existing in countries where advanced crustal deformation studies are carried out. Thus, INGV have co-located the classical seismological instrumentation (broad band seismometers and accelerometers) with GPS receivers to observe and quantify the whole seismic cycle. In this short paper, we describe the CGPS network, the technological choices for the monumentation and the data transmission, the data and metadata management and, finally, the data policy and the deliverables.INGVUnpublishedreserve

The RING network: improvements to a GPS velocity field in the central Mediterranean

Since 2004, a continuous Global Positioning System (GPS) network has been operated by the Istituto Nazionale di Geofisica e Vulcanologia (INGV) to investigate active tectonic processes in Italy and the surrounding regions, which are still largely debated. This important infrastructure is known as Rete Integrata Nazionale GPS (RING) network, and it consists of about 130 stations that are deployed all over Italy. The development and realization of a stable GPS monumentation, its integration with seismological instruments, and the choice of both satellite and internet data transmission, make this network one of the most innovative and reliable CGPS networks in the world. The technologically advanced development of the RING network has been accompanied by the development of different data processing strategies, which are mainly dependent on the use of different GPS analysis software. The different software-related solutions are here compared at different scales for this large network, and the consistency is evaluated and quantified within an RMS value of 0.3 mm/yr

Aeroacoustic study of a wavy stator leading edge in a realistic fan/OGV stage

International audienceThe effect of sinusoidal serrations applied to the leading-edge of the vanes of a realistic fan stage is investigated using high-fidelity numerical simulations. The CFD solver PowerFLOW based on a hybrid la ice-Boltzmann/very-large-eddy-simulation model is used to compute the unsteady flow and radiated noise of the-in source diagnostic test fan rig of the NASA Glenn Research Center. A computational model validated for three different geometries of the outlet guide vanes with straight leading edge is used. A subset of validation results is reported to prove the capability of the solver to accurately predict the influence of the stator geometry on the far-field noise. Different sinusoidal leading edge serrations are investigated for a radial and a swept stator and the same rotor and operating conditions. The influence of the serrations on the acoustic far-field and noise power level is reported in relation to the statistical properties of the velocity fluctuations in the wake of the rotor. Some noise reductions are obtained when the undulation amplitude and wavelength are large enough compared to the integral scales of the impinging turbulence fluctuations

Aeroacoustic study of a wavy stator leading edge in a realistic fan/OGV stage

The effect of sinusoidal serrations applied to the leading-edge of the vanes of a realistic fan stage is investigated using high-fidelity numerical simulations. The CFD solver PowerFLOW based on a hybrid lattice-Boltzmann/very-large-eddy-simulation model is used to compute the unsteady flow and radiated noise of the 22-in source diagnostic test fan rig of the NASA Glenn Research Center. A computational model validated for three different geometries of the outlet guide vanes with straight leading edge is used. A subset of validation results is reported to prove the capability of the solver to accurately predict the influence of the stator geometry on the far-field noise. Different sinusoidal leading edge serrations are investigated for a radial and a swept stator and the same rotor and operating conditions. The influence of the serrations on the acoustic far-field and noise power level is reported in relation to the statistical properties of the velocity fluctuations in the wake of the rotor. Some noise reductions are obtained when the undulation amplitude and wavelength are large enough compared to the integral scales of the impinging turbulence fluctuations.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Wind Energ

ASSOCIATION OF BEHAVIOURAL AND PHARMACOLOGICAL THERAPY IN AN AGGRESSIVE DOG

Abstract Dog aggression is a serious public health issue. More than 4 million dog bites to humans are estimated to occur each year, and up to 42% of dogs presented to behaviour clinics do so for aggression toward other dogs. Aggression places a serious strain on the human-animal bond. Dogs frequently are surrendered to shelters for behavioural reasons, including aggression. Several therapeutic approaches have been proposed, here, a combination of pharmacological and behavioural therapy has been used for the treatment of a severe case of aggressiveness in a dog. Extreme aggressive behaviour (general anxiety, fear aggression and possessive aggression) was diagnosed in a male pit bull aged 10 months. Two behaviour-therapy techniques (relaxation and counter-conditioning) were associated with a pharmacological therapy The pharmacological approach consisted in the per os administration of fluvoxamine as follows: 2 mg/kg/day every 12 hours for 30 days, 4 mg/kg/day for 150 days. The dose was then gradually reduced in 90 days. The behavioural therapy started after three weeks of drug administration. After 180 days no aggressive behaviour was detected anymore and the drug administration was gradually reduced in 90 days. The positive follow up suggests that behavioural therapy may be effective, by itself, in controlling aggressive temperaments but the combination with pharmacological treatment is often necessary to start applying behavioural techniques

GDNF-mediated rescue of the nigrostriatal system depends on the degree of degeneration

Glial cell-line derived neurotrophic factor (GDNF) is a promising therapeutic molecule to treat Parkinson’s disease. Despite an excellent profile in experimental settings, clinical trials testing GDNF have failed. One of the theories to explain these negative outcomes is that the clinical trials were done in late-stage patients that have advanced nigrostriatal degeneration and may therefore not respond to a neurotrophic factor therapy. Based on this idea, we tested if the stage of nigrostriatal degeneration is important for GDNF-based therapies. Lentiviral vectors expressing regulated GDNF were delivered to the striatum of rats to allow GDNF expression to be turned on either while the nigrostriatal system was degenerating or after the nigrostriatal system had been fully lesioned by 6-OHDA. In the group of animals where GDNF expression was on during degeneration, neurons were rescued and there was a reversal of motor deficits. Turning GDNF expression on after the nigrostriatal system was lesioned did not rescue neurons or reverse motor deficits. In fact, these animals were indistinguishable from the control groups. Our results suggest that GDNF can reverse motor deficits and nigrostriatal pathology despite an ongoing nigrostriatal degeneration, if there is still a sufficient number of remaining neurons to respond to therapy

Destabilizing Domains Enable Long-Term and Inert Regulation of GDNF Expression in the Brain

Regulation of therapeutic transgene expression can increase the safety of gene therapy interventions, especially when targeting critical organs such as the brain. Although several gene expression systems have been described, none of the current systems has the required safety profile for clinical applications. Our group has previously adapted a system for novel gene regulation based on the destabilizing domain degron technology to successfully regulate glial cell-line derived neurotrophic factor in the brain (GDNF-F-DD). In the present study, we used GDNF-F-DD as a proof-of-principle molecule to fully characterize DD regulation in the brain. Our results indicate that DD could be regulated in a dose-dependent manner. In addition, GDNF-F-DD could also be induced in vivo repeatedly, without loss of activity or efficacy in vivo. Finally, DD regulation was able to be sustained for 24 weeks without loss of expression or any overt toxicity. The present study shows that DD has great potential to regulate gene expression in the brain

Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling

The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5′ end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain

Deconvolution of spatial sequencing provides accurate characterization of hESC-derived DA transplants in vivo

Cell therapy for Parkinson’s disease has experienced substantial growth in the past decades with several ongoing clinical trials. Despite increasing refinement of differentiation protocols and standardization of the transplanted neural precursors, the transcriptomic analysis of cells in the transplant after its full maturation in vivo has not been thoroughly investigated. Here, we present spatial transcriptomics analysis of fully differentiated grafts in their host tissue. Unlike earlier transcriptomics analyses using single-cell technologies, we observe that cells derived from human embryonic stem cells (hESCs) in the grafts adopt mature dopaminergic signatures. We show that the presence of phenotypic dopaminergic genes, which were found to be differentially expressed in the transplants, is concentrated toward the edges of the grafts, in agreement with the immunohistochemical analyses. Deconvolution shows dopamine neurons being the dominating cell type in many features beneath the graft area. These findings further support the preferred environmental niche of TH-positive cells and confirm their dopaminergic phenotype through the presence of multiple dopaminergic markers