Cross-linking cellular prion protein induces neuronal type 2-like hypersensitivity

Background: Previous reports identified proteins associated with ‘apoptosis’ following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with antiPrP antibody-mediated ‘apoptosis’, however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response. Methods: Initially, we predicted the allergenicity of the epitope sequences associated with ‘neurotoxic’ anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of mouse primary neurons (MPN), neuroblastoma cells (N2a) and microglia (N11) cell lines lead to a neuronal allergenic response. Results: In-Silico studies showed that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for previously reported ‘neurotoxic’ antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of antiPrPC antibodies on N2a cells, we identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, we identified 8 neuronal allergenic-related proteins following treatment of N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells when compared with untreated cells. Antibody treatment of MPN or MPN co-cultured with antibody-treated N11 led to identifying 10 and 7 allergenic-related proteins when compared with untreated cells. However, comparison with 3F4 antibody treatment revealed 5 and 4 allergenic-related proteins respectively. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Finally, co-culture of N2a or MPN with N11-treated with anti-PrP antibodies resulted in significant accumulation of NO and IL6 but not TNF-a in the cell culture media supernatant. Conclusions: This study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal hypersensitivity response and highlights the important role of microglia in triggering an IgG-mediated neuronal hypersensitivity response. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative disorders to derive safe and targeted biotherapeutics

Frequency steerable acoustic transducers

Structural health monitoring (SHM) is an active research area devoted to the assessment of the structural integrity of critical components of aerospace, civil and mechanical systems. Guided wave methods have been proposed for SHM of plate-like structures using permanently attached piezoelectric transducers, which generate and sense waves to evaluate the presence of damage. Effective interrogation of structural health is often facilitated by sensors and actuators with the ability to perform directional scanning. In this research, the novel class of Frequency Steerable Acoustic Transducers (FSATs) is proposed for directional generation/sensing of guided waves. The FSATs are characterized by a spatial arrangement of the piezoelectric material which leads to frequency-dependent directionality. The resulting FSATs can be employed both for directional sensing and generation of guided waves, without relying on phasing and control of a large number of channels. Because there is no need for individual control of transducer elements, hardware and power requirements are drastically reduced so that cost and hardware limitations of traditional phased arrays can be partially overcome. The FSATs can be also good candidates for remote sensing and actuation applications, due to their hardware simplicity and robustness. Validation of the proposed concepts first employs numerical methods. Next, the prototyping of the FSATs allows an experimental investigation confirming the analytical and numerical predictions. Imaging algorithm based on frequency warping is also proposed to enhance results representation.PhDCommittee Chair: Ruzzene, Massimo; Committee Member: Ahuja, Krish; Committee Member: Degertekin, Levent; Committee Member: Lieuwen, Tim; Committee Member: Michaels, Jennife

Behavioural characterization of M1000 prion strain pathogenesis and the acute neurotoxicity of disease-associated prion protein in vivo

© 2015 Dr. Matteo SenesiPrion diseases are a group of transmissible, fatal, neurodegenerative diseases naturally afflicting a number of mammals, including humans and no effective treatments currently exist. The most common human prion disease phenotype is Creutzfeldt-Jakob disease (CJD), which is quite rare, occurring at a rate of around 1-2 per million per year, with other forms of human prion disease such as iatrogenic CJD, Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) much less common. Kuru, as well, a prion disease endemic to the Fore linguistic group in the Eastern Highlands of New Guinea, is now most likely extinct stemming from the effective outlawing of cannibalistic mourning rituals amongst these natives. Although rare, concerns around inter- and intra-species transmissibility generally give prion diseases a heightened profile, especially when dealing with health care settings. The central pathogenic event in prion disease is understood to be the misfolding of the normal or cellular form of the prion protein (PrPc) into a β-strand enriched, disease-causing conformer (PrPSc), which characteristically manifests altered biochemical properties such as detergent insolubility, relative protease resistance and heightened tendency to aggregate. The refolding can be promoted by genetic mutations in the gene encoding PrPc (PRNP) associated with genetic prion disease or occur through a stochastic misfolding event (sporadic prion disease). Once present (including through exogenous introduction), PrPSc propagation proceeds through an auto-catalytic self-templating mechanism, wherein PrPc forms a hetero-dimer with a molecule of PrPSc. The normal function(s) of the glycoprotein PrPc remain unresolved but the protein is ubiquitously expressed with highest levels found in neurons and after a number of post-translational modifications is normally bound through a glycosylphosphatidylinositol anchor to the external cellular surface within lipid rafts. Prions, the infectious unit of prion diseases, are believed to be composed predominantly or exclusively by PrPSc, with host tissue expression of PrPc absolutely essential for successful disease transmission; current evidence suggests that different species of PrPSc may underpin neurotoxicity and transmissibility. The ongoing imperative to develop effective treatments has been pari passu with investigations to better understand the neurotoxic events and pathogenic mechanisms sub-serving prion disease, with the belief that improved mechanistic insights will facilitate development of effective therapies. Of additional interest has been the recent parallel investigations of other common neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, which have identified “prion-like” mechanisms of misfolded protein propagation (through self-templating to generate multimers including soluble oligomers and amyloid fibrils), as well as the inter-cellular spreading mechanisms of misfolded proteins. Consequently, an improved understanding of the mechanisms of prion disease neurotoxicity might offer broader insights in the field of neurodegenerative disease pathogenesis. Although in vitro models of pathogenesis and neurotoxicity can and have offered useful and worthwhile mechanistic insights, in vivo models are arguably more valid and while many excellent long-term in vivo models of prion disease exist, there is a noteworthy paucity of acute, in vivo, prion neurotoxicity models to assist delineation of neurotoxic mechanisms and the PrPSc species responsible for neuronal dysfunction and death. Several studies previously attempted to investigate prion neurotoxic mechanisms with discordant results; in particular between the uses of ex vivo prions and refolded synthetic PrP molecules. While ex vivo prions obtained from inoculated mice did not show any apparent acute neurotoxicity or inflammatory reaction based on morphological changes (compared to controls) before de novo prion propagation occurred, synthetic and recombinant PrP peptides, often enriched in β-strand secondary structure (crudely recapitulating some features of PrPSc) were able to induce noticeable neuronal shrinkage, increased reactive oxygen species and astrocytic morphological changes in vivo and in vitro days or weeks after exposure, even in PrP-deficient models. This stands in contrast to how ex vivo prions can be easily serially passaged once adapted in a certain species readily inducing prion disease in in vivo models. The discrepancies in acute neurotoxic effects observed between the use of ex vivo prions versus PrP peptides obtained with different methods highlights the necessity to clarify whether such differences relate to biologically invalid or artefactual toxicity or the existence of qualitative differences between PrPSc species addressing the enigma of potential prion species with enhanced neurotoxicity and others with mostly infectious properties. To try to further fundamental insights into genuine prion neurotoxicity and in particular to try to facilitate the capacity to delineate directly neurotoxic PrPSc species, I developed a novel, acute, in vivo model of prion neurotoxicity, utilising bona fide prions harboured in ex vivo preparations. With the use of the M1000 prion strain, serially passaged in wild type Balb/c and C57BL/6 mice, extensively studied in my laboratory and shown to selectively target the hippocampus early in disease evolution, I characterized the natural evolution of the behavioural (ethological and cognitive) disturbances following routine intra-cerebral inoculation of prions in this in vivo wild type mouse model. It is noteworthy in the M1000 model that there is an “eclipse period” immediately following routine intra-cerebral inoculation that lasts up to day 21 post-inoculation during which minimal net prion propagation appears to be occurring. Through these studies I determined those behavioural testing paradigms centred on hippocampal function which were able to detect ethological and cognitive disturbances, finding an early, progressive deleterious effect of prion propagation on retrieval of spatial and recognition memory (Y maze) followed by impairment in spatial learning (Barnes maze) and progressive cognitive alteration of conditioned fear memory extinction prior to any significant motoric impairment detected through Rotarod assessments. These results demonstrated novel differences between stages of disease incubation with subtle and progressive cognitive impairments never reported before in prion disease in vivo models. After extensive preliminary testing to develop the surgical technique, mainly utilising cellular fractions derived from non-infected and chronically M1000 infected RK13 cells for comparisons to naïve mice, I then incorporated those behavioural tests shown to be sensitive to natural prion disease evolution as part of a novel acute model to evaluate prion neurotoxicity employing stereotactic surgery to intra-cranially inject M1000 prions into wild-type C57BL/6 mice and transgenic PrP-knockout mice (PrP0/0) back-crossed to C57BL/6 wild type mice for 10 generations. Under ketamine/xylazine anaesthesia, 2 μl of 10% brain homogenates (w/v in PBS) from mice dying of terminal prion disease (M1000 mice) or from age/sex matched mice previously sham inoculated with normal brain homogenate (control mice) was stereotactically injected into each CA1 region at 400nl/minute (bregma coordinates: - 2.5mm; ± 2.5mm; and depth 1.5mm). Assessments utilising Coomassie Brilliant blue staining of control brain homogenates confirmed accuracy of the injection location and a broad anatomical dispersion of the inoculum along the entire hippocampus. Starting day 7 post-injection, groups of M1000 and control mice (~n=10 per group) were interrogated with the behavioural assessment battery centred on hippocampal-dependent tasks, which included Open Field testing, Burrowing, Y maze, Barnes maze and conditioned fear memory (using tone-foot shock pairing) providing insights into spatial memory and learning, recognition memory and conditioned fear memory acquisition and extinction. My results derived from this new model showed for the first time acute behavioural disturbances across a number of behavioural assessments supporting direct or intrinsic neurotoxic properties of ex vivo prions in wild-type mice in the absence of any gross motor disturbance. In particular, the hippocampal damage observed is limited to a selective delayed retrieval of spatial memory without a significant impairment in spatial learning. This is consistent with other findings related to experimental hippocampal lesions wherein partial ventral lesions impact significantly retrieval but allow spatial learning comparable to controls. The disturbance achieved with a single bilateral inoculation of M1000 prions also facilitates fear memory extinction 48 hours after conditioning but does not alter contextual fear memory. Importantly, PrP0/0 mice did not display any acute deleterious effects in conditioned fear memory extinction suggesting an apparent immunity to the toxicity observed in wild type mice using this paradigm and suggesting possible involvement of endogenous PrPc in the acute prion neurotoxic signalling pathway. Other biochemical insights derived from brain samples of M1000 wild type mice suggest a possible selective astrocytic vulnerability to acute prion toxicity without significant neuronal involvement, inflammatory response or cellular apoptosis when compared to controls. In conclusion, my PhD studies have developed and partly validated a novel, acute, in vivo, neurotoxicity model utilising stereotaxic surgery for delivery of ex vivo prion containing preparations. Future studies are needed to complete the validation of this model, including studies employing ex vivo prion preparations depleted and enriched in PrPSc, as well as detailed hippocampal morphological assessments of pre- and post-synaptic integrity (especially in the stratum oriens and radiatum), astrocytic and microglial changes, neuronal loss (in the stratum pyramidale) and the presence of activation of apoptosis. As such, further elaboration and validation of this model should create a valuable tool to facilitate characterization of the mechanisms underlying PrPSc synaptic and neuronal toxicity, as well as support parallel biophysical studies aimed at delineating the PrPSc species directly responsible for synaptic and neuronal dysfunction

Experimental demonstration of directional GW generation through wavenumber-spiral Frequency Steerable Acoustic Actuators

Directional inspection using guided waves (GWs) is a convenient approach for Structural Health Monitoring (SHM) of large 2D regions. While beam steering is conventionally achieved through phased arrays, often at the cost of a considerable hardware and software complexity, a single, differential-channel actuator is employed in this work to send GWs in arbitrary directions by properly choosing the excitation frequency. This frequency-steerable directional scanning is achieved through an angle-dependent wavelength tuning provided by the peculiar shape of the piezoelectric patch which forms the transducer. Although the underlying theoretical framework of Frequency-Steerable Acoustic Transducers (FSATs) encompasses both directional generation and sensing of GWs, directional actuation has only been predicted by simulations so far. This paper presents for the first time an experimental study of directional GW generation on an aluminum plate through an FSAT fabricated by inkjet printing of the electrodes on a PVDF substrate and previously operated in sensing mode

Fabrication and Characterization of a Wavenumber-Spiral Frequency-Steerable Acoustic Transducer for Source Localization in Plate Structures

This paper reports on the fabrication and the experimental characterization of a wavenumber frequency-steerable acoustic transducer (WS-FSAT). Here, the transducer is employed for the localization of broadband acoustic events corresponding to the propagation of guided elastic waves in an isotropic plate. The WS-FSAT records the plate response and defines the source location through a time-frequency analysis of the received signal. This is achieved by exploiting the frequency selective response of the transducer which directly maps the dominant component of the received signal to the direction of arrival of the incoming wave. This feature is the result of the spatial filtering effect produced by the characteristic shape of the sensing surface, which is designed in the wavenumber domain. Experiments are performed on a prototype fabricated on a polyvinylidene fluoride substrate mounted on an aluminum test plate. Tests are conducted for various source locations, and with multiple sources activated simultaneously. The results highlight the robustness of the proposed device, its good sensitivity and angular resolution, as well as the low complexity of hardware and signal processing. This paper suggests the WS-FSAT as an attractive solution for the detection of broadband acoustic events, such as impacts on structural substrates, and its potential use as part of active structural health monitoring systems based on pitch-catch or pulse-echo operations

Salvage of advanced carpal collapse: proximal row carpectomy with pyrocarbon resurfacing of the capitate versus four-corner arthrodesis

In this retrospective study we report on two comparable groups of patients with advanced carpal arthritis treated with either proximal row carpectomy combined with a pyrocarbon resurfacing of the capitate (31 patients) or a four-corner arthrodesis and dorsal plating (26 patients). Follow-up time was 46 months (24-118). Except for a modestly higher radial wrist deviation in the patients treated with four-corner arthrodesis, there were no significant differences in outcomes between the groups. Asymptomatic progression of osteoarthritis in the lunate fossa was observed in four cases in both groups. Two cases were converted to a total wrist arthrodesis in the pyrocarbon group compared with one case in the four-corner arthrodesis group. Although four-corner arthrodesis remains the reference standard in the treatment of wrist osteoarthritis with involvement of the midcarpal joint, proximal row carpectomy combined with pyrocarbon resurfacing of the capitate is an alternative option. It can even be used in selected cases with erosion of the lunate fossa

Hydrogen detection limits and instrument sensitivity of high-resolution broadband neutron spectrometers

The limits of detection (LOD) and quantitation (LOQ) in the mass domain, for broadband vibrational spectroscopy with neutrons on the TOSCA spectrometer at the ISIS Pulsed Neutron and Muon Source (UK), have been studied. The well-known 3σ and 10σ approaches are used through a specifically developed analytical procedure that is based on the calculation of the integrated spectral intensities in selected energy-transfer ranges, as a function of mass of standard reference materials and calibrants, such as ZrH2, 2,5-diiodothiophene, and low-density polyethylene. The analysis shows that the blank, that is, the instrument setup without the analyte, plays a critical role in the measurement performance, especially for small specimen quantities. The results point that TOSCA enables detection of 128 μmol (LODH) and quantitation of 428 μmol (LOQH) of elemental hydrogen analytes in ZrH2. The determined values for this and other standards allow for the assessment of the calibration curve design and instrument sensitivity and define a method to be used for inelastic neutron scattering spectrometers such as TOSCA, or VESPA, the new beamline under construction at the European Spallation Source in Lund (Sweden)

Tailored behavioural tests reveal early and progressive cognitive deficits in M1000 prion disease

Prion diseases are pathogenically linked to the normal cellular prion protein (PrPC) misfolding into abnormal conformers (PrPSc), with PrPSc accumulation underpinning both transmission and neurotoxicity. Despite achieving this canonical understanding, however fundamental questions remain incompletely resolved, including the level of pathophysiological overlap between neurotoxic and transmitting species of PrPSc and the temporal profiles of their propagation. To further investigate the likely time of occurrence of significant levels of neurotoxic species during prion disease development, the well characterised in vivo M1000 murine model was employed. Following intracerebral inoculation, detailed serial cognitive and ethological testing at specified time points suggested subtle transition to early symptomatic disease from ∼50% of the overall disease course. In addition to observing a chronological order for impaired behaviours, different behavioural tests also showed distinctive profiles of evolving cognitive impairments with the Barnes maze demonstrating a relatively simple linear worsening of spatial learning and memory over an extended period while in contrast a conditioned fear memory paradigm previously untested in murine prion disease demonstrated more complex alterations during disease progression. These observations support the likely production of neurotoxic PrPSc from at least just prior to the mid-point of murine M1000 prion disease and illustrate the likely need to tailor the types of behavioural testing across the time course of disease progression for optimal detection of cognitive deficits