Exploring the aetiology of pre-motor Parkinson's disease and the efficacy of potential neuroprotective therapies

The present thesis aimed at providing insight into the aetiology and treatment of Parkinson’s disease (PD), which symptomatology consists of both motor and non-motor symptoms (NMSs). The latter have been linked to a loss of neurotransmitters other than dopamine and they have been shown to be modulated by treatments that do not act directly on the dopaminergic system, such as the glucagon-like peptide-1 receptor agonist exendin-4 (EX-4). Nevertheless, the aetiology of NMs, alongside with their potential treatments, has yet to be fully investigated. In this study, through injections of the neurotoxins N-N-ethyl-2-bromobenzylamine (DSP-4) and 6-hydroxydopamine (6-OHDA), a rat model of early-stage PD was developed and validated. Animals displayed the NMS hyposmia and memory impairments in the absence of motor symptoms, suggesting the PD model is representative of an early stage of the disease. Next, the effect of partial noradrenergic and dopaminergic denervation in several brain regions within the olfactory pathway was investigated using immunohistochemical techniques. Surprisingly, the combined denervation led to a reduction in the expression of interneuronal calcium binding proteins (CBPs) in the primary olfactory cortex and prefrontal cortex, whilst the expression in the olfactory bulbs was found to be increased, alongside with dopaminergic expression. Additionally, GABAergic cells in CA2 of the PD model were found to be decreased compare with controls. Interestingly, the observed structural changes were partially prevented following treatment with EX-4. Additionally, two preliminary studies were conducted using the early-stage PD model to test two potential new treatments, a novel viral vector and probiotics, and their effectiveness in preventing neuronal loss in the Substantia Nigra. Overall, this rat model of early-stage PD offers a useful means for research into early diagnosis as well as early intervention of PD, possibly resulting in a delay of disease progression together with improved patient’s quality of life

Protein Deimination Signatures in Plasma and Plasma-EVs and Protein Deimination in the Brain Vasculature in a Rat Model of Pre-Motor Parkinson’s Disease

The identification of biomarkers for early diagnosis of Parkinson’s disease (PD) is of pivotal importance for improving approaches for clinical intervention. The use of translatable animal models of pre-motor PD therefore offers optimal opportunities for novel biomarker discovery in vivo. Peptidylarginine deiminases (PADs) are a family of calcium-activated enzymes that contribute to protein misfolding through post-translational deimination of arginine to citrulline. Furthermore, PADs are an active regulator of extracellular vesicle (EV) release. Both protein deimination and extracellular vesicles (EVs) are gaining increased attention in relation to neurodegenerative diseases, including in PD, while roles in pre-motor PD have yet to be investigated. The current study aimed at identifying protein candidates of deimination in plasma and plasma-EVs in a rat model of pre-motor PD, to assess putative contributions of such post-translational changes in the early stages of disease. EV-cargo was further assessed for deiminated proteins as well as three key micro-RNAs known to contribute to inflammation and hypoxia (miR21, miR155, and miR210) and also associated with PD. Overall, there was a significant increase in circulating plasma EVs in the PD model compared with sham animals and inflammatory and hypoxia related microRNAs were significantly increased in plasma-EVs of the pre-motor PD model. A significantly higher number of protein candidates were deiminated in the pre-motor PD model plasma and plasma-EVs, compared with those in the sham animals. KEGG (Kyoto encyclopedia of genes and genomes) pathways identified for deiminated proteins in the pre-motor PD model were linked to “Alzheimer’s disease”, “PD”, “Huntington’s disease”, “prion diseases”, as well as for “oxidative phosphorylation”, “thermogenesis”, “metabolic pathways”, “Staphylococcus aureus infection”, gap junction, “platelet activation”, “apelin signalling”, “retrograde endocannabinoid signalling”, “systemic lupus erythematosus”, and “non-alcoholic fatty liver disease”. Furthermore, PD brains showed significantly increased staining for total deiminated proteins in the brain vasculature in cortex and hippocampus, as well as increased immunodetection of deiminated histone H3 in dentate gyrus and cortex. Our findings identify EVs and post-translational protein deimination as novel biomarkers in early pre-motor stages of PD

Single prazosin infusion in prelimbic cortex Fosters extinction of amphetamine-induced conditioned place preference

Exposure to drug-associated cues to induce extinction is a useful strategy to contrast cue-induced drug seeking. Norepinephrine (NE) transmission in medial prefrontal cortex has a role in the acquisition and extinction of conditioned place preference induced by amphetamine. We have reported recently that NE in prelimbic cortex delays extinction of amphetamine-induced conditioned place preference (CPP). A potential involvement of α1-adrenergic receptors in the extinction of appetitive conditioned response has been also suggested, although their role in prelimbic cortex has not been yet fully investigated. Here, we investigated the effects of the α1-adrenergic receptor antagonist prazosin infusion in the prelimbic cortex of C57BL/6J mice on expression and extinction of amphetamine-induced CPP. Acute prelimbic prazosin did not affect expression of amphetamine-induced CPP on the day of infusion, while in subsequent days it produced a clear-cut advance of extinction of preference for the compartment previously paired with amphetamine (Conditioned stimulus, CS). Moreover, prazosin-treated mice that had extinguished CS preference showed increased mRNA expression of brain-derived neurotrophic factor (BDNF) and post-synaptic density 95 (PSD-95) in the nucleus accumbens shell or core, respectively, thus suggesting that prelimbic α1-adrenergic receptor blockade triggers neural adaptations in subcortical areas that could contribute to the extinction of cue-induced drug-seeking behavior. These results show that the pharmacological blockade of α1-adrenergic receptors in prelimbic cortex by a single infusion is able to induce extinction of amphetamine-induced CPP long before control (vehicle) animals, an effect depending on contingent exposure to retrieval, since if infused far from or after reactivation it did not affect preference. Moreover, they suggest strongly that the behavioral effects depend on post-treatment neuroplasticity changes in corticolimbic network, triggered by a possible “priming” effect of prazosin, and point to a potential therapeutic power of the antagonist for maladaptive memories

Effects of a probiotic suspension Symprove™ on a rat early-stage Parkinson’s disease model

An increasing number of studies in recent years have focused on the role that the gut may play in Parkinson’s Disease (PD) pathogenesis, suggesting that the maintenance of a healthy gut may lead to potential treatments of the disease. The health of microbiota has been shown to be directly associated with parameters that play a potential role in PD including gut barrier integrity, immunity, function, metabolism and the correct functioning of the gut-brain axis. The gut microbiota (GM) may therefore be employed as valuable indicators for early diagnosis of PD and potential targets for preventing or treating PD symptoms. Preserving the gut homeostasis using probiotics may therefore lead to a promising treatment strategy due to their known benefits in improving constipation, motor impairments, inflammation, and neurodegeneration. However, the mechanisms underlying the effects of probiotics in PD are yet to be clarified. In this project, we have tested the efficacy of an oral probiotic suspension, Symprove™, on an established animal model of PD. Symprove™, unlike many commercially available probiotics, has been shown to be resistant to gastric acidity, improve symptoms in gastrointestinal diseases and improve gut integrity in an in vitro PD model. In this study, we used an early-stage PD rat model to determine the effect of Symprove™ on neurodegeneration and neuroinflammation in the brain and on plasma cytokine levels, GM composition and short chain fatty acid (SCFA) release. Symprove™ was shown to significantly influence both the gut and brain of the PD model. It preserved the gut integrity in the PD model, reduced plasma inflammatory markers and changed microbiota composition. The treatment also prevented the reduction in SCFAs and striatal inflammation and prevented tyrosine hydroxylase (TH)-positive cell loss by 17% compared to that observed in animals treated with placebo. We conclude that Symprove™ treatment may have a positive influence on the symptomology of early-stage PD with obvious implications for the improvement of gut integrity and possibly delaying/preventing the onset of neuroinflammation and neurodegeneration in human PD patients

GAMMA: An 8-channel High Dynamic Range ASIC for SiPM-Based Readout of Large Scintillators

LaBr 3 crystals are well known to provide the brightest light pulses among commercially available scintillators. Recent developments in SiPMs brought to the market low-darkcount, high-PDE photodetectors that can offer high dynamic range readout of LaBr 3 . While state-of-the-art front-end electronics is suitable for standard dynamic range applications, e.g. for PET, experiments needing large DR require novel solutions. Our project aims at developing a wide DR (100 keV - 20 MeV) gamma spectrometer and imager with state-of-the-art energy resolution. In order to design a specific front-end for the application, the signal range impinging on every different SiPM was simulated and measured. Results of preliminary measurements showed that higher the energy impinging the crystal higher is the signal spread among the channels. The charge spread was then estimated to be 3 pC - 3nC, to be acquired without any gain change during the acquisition. The input stage was designed to read the current and scale it down to be processed by the following filter. The timevariant filter was designed to act as a self-triggered gated integrator. In order to exploit all the estimated charge range it was implemented a signal dependent Active Gain Control (AGC): the gain is proportionally reduced in order to increase the saturation charge to 2.5 nC. Measurements were performed in order to demonstrate the working principle of the AGC, verify the resulting dynamic range and the crosstalk between channels. 80 dB DR was measured and AGC proved its functionality. Experimental measurements were also performed in order to verify spectroscopy resolution: 3.8% at 622 keV was measured at room temperature