<p>Parkinson’s disease (PD) is an age-related neurodegenerative disorder characterized by pathological features that include the selective loss of dopaminergic (DA) neurons in the substantia nigra (SN) region of the midbrain and the presence of intraneuronal Lewy body and Lewy neurite inclusions primarily comprised of fibrillar forms of the pre-synaptic protein alpha-synuclein (aSyn).  aSyn aggregation has been implicated as a critical event in PD pathogenesis, and mutant forms of aSyn are associated with familial, early-onset forms of the disease. PD presents clinically as a movement disorder through appearance of its cardinal motor symptoms of bradykinesia, rigidity, postural instability, and resting tremor. However, upon manifestation of these clinical symptoms, over 50% of the DA nigral neurons have been lost, suggesting that PD neuropathology likely begins 10-15 years prior to the clinical onset. Current PD treatment is focused on symptomatic therapy through dopamine replacement strategies, and there are currently no medications available to prevent or slow disease progression. </p> <p>A major hurdle to developing disease-modifying therapies for PD is a lack of knowledge of the molecular phenomena responsible for the death of DA neurons in the SN. Multiple cellular pathways that are dysregulated in PD include protein clearance systems and oxidative stress responses. Changes in these systems by environmental or genetic perturbations cause increased aSyn aggregation, which in turn leads to increased oxidative and proteasomal stress, thereby generating a vicious cycle culminating in the death of nigral DA neurons. Therefore, strategies to activate these protective pathways have the potential to reduce aSyn aggregation and halt neurodegeneration. One such mechanism is through the activation of the stress-induced transcription factor, Nfe2L1. Nfe2L1 is a cap ‘n’ collar basic leucine zipper (CNC-bZIP) transcription factor that forms heterodimers with small Maf proteins. In turn, the heterodimers bind to antioxidant response element (ARE) sequences in the promoter regions of cytoprotective genes, such as genes encoding proteasome subunits and proteins involved in the glutathione synthesis pathway. In the studies summarized in Chapter 2, we have characterized a pre-clinical <em>in vivo</em> model of PD involving aSyn overexpression in rat SN and used this model to investigate whether Nfe2L1 co-expression could alleviate aSyn neurotoxicity by reducing cytosolic aSyn levels via proteasome activation. Using unbiased stereology to determine nigral DA neuron cell counts, we found that Nfe2L1 may have a protective effect against aSyn-mediated nigral DA neurodegeneration. Surprisingly, we observed no increase in proteasome subunit expression through quantitative PCR or immunoblotting. However, by using a single-neuron analysis approach, we observed a significant increase in PSMC1 subunit expression, suggesting that Nfe2L1 expression could indeed lead to an upregulation of proteasome subunits and an increase in proteasome function. Future experiments will be aimed at determining whether Nfe2L1 expression results in an increase in proteasome activity, an enhancement of aSyn degradation, and a decrease in the burden of proteinase K-resistant (Lewy-like) aSyn aggregates in rat SN. </p> <p>The ability to detect PD in the pre-symptomatic stage is necessary for the development of novel therapies to enable treatment prior to irreversible neuronal loss. Biomarkers with high sensitivity and specificity are critical for early PD detection. aSyn levels have been measured in human biofluids, such as blood and CSF, as a potential biomarker for PD diagnosis and for monitoring disease progression. aSyn can undergo a number of post-translational modifications (PTMs), and a particular form of the protein phosphorylated at serine 129 (pS129-aSyn) is enriched in Lewy bodies, making it an attractive candidate for biomarker studies. Although there are several antibodies targeting pS129, little is known about the influence of PTMs close to pS129 on the antibodies’ affinity for the pS129 epitope, or how these neighboring PTMs could affect assays developed to quantify pS129-aSyn in biofluids. In the studies summarized in Chapter 3, we characterized the impact of PTMs near pS129 on the affinity of currently available pS129-aSyn antibodies for their target antigen using biolayer interferometry (BLI). BLI analysis of the D1R1R pS129-aSyn antibody (Cell Signaling Technology) revealed that tyrosine phosphorylation or nitration at Y133 greatly reduced antibody affinity. In contrast, the MJF-R13 pS129-aSyn antibody (Abcam) was found to have reduced affinity for peptide targets nitrated or phosphorylated on Y125 or phosphorylated on Y133. </p> <p>While aSyn is typically investigated as it relates to PD and other neurodegenerative disorders, recently reported evidence suggests that aSyn downregulation could be linked to an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD).  ASD is a complex neurodevelopmental disorder that presents with characteristic behavioral symptoms of social and communication impairments and restricted, repetitive behaviors. Diagnosis of ASD is based on the presentation of these behavioral symptoms, typically appearing at ~12 months of age, yet individuals don’t receive a diagnosis until the age of five. Early identification and early treatment are regarded as two of the most important factors for improving patient outcomes. SNCA gene deletions and loss-of-function duplications have been found in individuals with intellectual disability, developmental delay, and/or ASD, leading to the idea that reduced aSyn expression may be a biomarker for early ASD diagnosis and may play a role in ASD neuronal dysfunction. In the studies summarized in Chapter 4, we evaluated salivary aSyn as a potential biomarker for early ASD diagnosis and examined SNCA-/- iPSC-derived cortical neurons for indications of ASD-related neuronal anomalies. Our preliminary results suggest that salivary aSyn is reduced in individuals with neurodevelopmental disorders such as ASD and Fragile X Syndrome (FXS), a result consistent with findings of reduced aSyn in serum and plasma from ASD individuals. Furthermore, SNCA-/- iPSC-derived cortical neurons depleted of aSyn expression had increased soma size, a characteristic of iPSC-derived cortical neurons with ASD-associated mutations in the genes encoding MECP2 and TSC1/2 These results suggest that SNCA gene disruptions play a role in ASD-related neuronal anomalies and dysfunction. </p> <p>Overall, the results presented in this thesis support a role for targeting aSyn protein expression in neurodegenerative and neurodevelopmental disorders, and they underscore the importance of designing aSyn biomarker immunoassays that faithfully report on each of these syndromes. Our data suggesting that Nfe2L1 could protect against nigral neurodegeneration by stimulating proteasome-mediated aSyn clearance imply that strategies to increase Nfe2L1-dependent transcriptional activity (e.g., using small molecule activators or gene therapy) could ameliorate pathological aspects of PD. The results presented here also highlight the need for sensitive and specific biomarker assays targeting multiple aSyn proteoforms, and they suggest that aSyn could be a viable biomarker for early ASD diagnosis. Finally, our findings provide the first evidence that aSyn down-regulation contributes to neuronal anomalies associated with ASD, in turn suggesting that strategies to increase cytosolic aSyn by preventing its degradation or through gene therapy could potentially mitigate ASD neuronal dysfunction. </p>

A doença de Parkinson (DP) caracteriza-se pela perda progressiva de neurônios dopaminérgicos da substância nigra, o que acarreta diversas disfunções motoras. Não há ainda tratamentos capazes de deter ou retardar a degeneração dos neurônios dopaminérgicos, e os medicamentos hoje empregados na clínica apenas amenizam os sintomas, sem alterar a progressão da DP. No presente estudo foi avaliada a atividade neuroprotetora do éster fenetil do ácido cafeico (CAPE), um componente abundante do própolis de abelhas, com atividade anti-inflamatória, antiviral, antioxidante e imunomodulatória. Estudos têm sugerido seus efeitos benéficos contra as doenças neurodegenerativas, incluindo a doença de Parkinson, e alguns mecanismos têm sido propostos; porém muitos dos estudos com CAPE foram feitos apenas em culturas celulares. Este é o primeiro estudo a demonstrar que a administração intraperitoneal do CAPE protege contra a perda neuronal dopaminérgica e a disfunção motora induzidas pela neurotoxina 6-OHDA em ratos, confirmando a capacidade do CAPE de atravessar a barreira hematoencefálica e exercer seus efeitos benéficos no sitema nervoso central (SNC). Adicionalmente foram empregados dois modelos in vitro para o delineamento de possíveis mecanismos de neuroproteção: (i) mitocôndrias isoladas de cérebro de ratos não tratados e (ii) células SH-SY5Y tratadas com 6-OHDA. Os achados in vivo e in vitro sugerem o envolvimento dos seguintes mecanismos: (i) atividade antioxidante (sequestro de ERO, neutralização de radicais livres e quelação de metais); (ii) atividade anti-inflamatória (inibição da ativação do NF-kB, TNF-?, IkK? e Ikk?); (iii) aumento da expressão da conexina 43; (iv) inibição da Transição de Permeabilidade Mitocondrial (TPM); (v) inibição da liberação de citocromo c e (vi) inibição da ativação da caspase-3, executora final da apoptose. Além disso, o estudo também demonstrou que, por si só, o CAPE não interfere nas funções mitocondriais, o que representa uma vantagem com relação a outros inibidores da TPM. Assim, de acordo com nossos achados, o CAPE é um agente neuroprotetor promissor e pode auxiliar em futuras estratégias terapêuticas para as doenças neurodegenerativas, bem como para o melhor entendimento dos mecanismos responsáveis pelo desenvolvimento dessas doenças. / Parkinson\'s disease (PD) is characterized by progressive loss of dopaminergic neurons in the substantia nigra, which causes various motor dysfunctions. There are no treatments able to delay or stop the degeneration of dopaminergic neurons and the therapy employed nowadays only alleviate the symptoms without altering the progression of PD. In the present study we evaluated the neuroprotective activity of caffeic acid phenethyl ester (CAPE), a compound abundant in honeybees\' propolis, with anti-inflammatory, antiviral, antioxidant and immunomodulatory activities. Its beneficial effects against neurodegenerative diseases, including Parkinson\'s disease, have been suggested and some mechanisms have been proposed; however, many of the studies with CAPE have been performed only in cell cultures. This is the first study to demonstrate that the intraperitoneal administration of CAPE protects against the dopaminergic neuronal loss and the motor dysfunction induced by the neurotoxin 6-OHDA in rats, confirming the ability of CAPE to cross the blood brain barrier and exert its beneficial effects on the central nervous system (CNS). Additionally two in vitro models were used to delineate the possible mechanisms of neuroprotection: (i) mitochondria isolated from the brain of non-treated rats and (ii) cells SH-SY5Y treated with 6-OHDA. The in vivo and in vitro findings suggest the involvement of the following mechanisms: (i) antioxidant activity (scavenger of ROS, free radicals neutralization and metal chelation); (ii) anti-inflammatory activity (inhibition of activation of NF-kB, TNF-?, IkK? and Ikk?); (iii) increased expression of connexin-43; (iv) Inhibition of Mitochondrial Permeability Transition (MPT); (v) inhibition of cytochrome c release (vi) inhibition of the activation of caspase-3, the final executioner of apoptosis. Furthermore, the study also showed that CAPE alone does not interfere with mitochondrial functions, which represents an advantage in relation to other inhibitors of MPT. Therefore, according to our findings, CAPE is a promising neuroprotective agent and may be useful for future therapeutic strategies for neurodegenerative diseases, as well as to better understand the mechanisms responsible for the development of these diseases.

A doença de Parkinson (DP) caracteriza-se pela perda progressiva de neurônios dopaminérgicos da substância nigra, o que acarreta diversas disfunções motoras. Não há ainda tratamentos capazes de deter ou retardar a degeneração dos neurônios dopaminérgicos, e os medicamentos hoje empregados na clínica apenas amenizam os sintomas, sem alterar a progressão da DP. No presente estudo foi avaliada a atividade neuroprotetora do éster fenetil do ácido cafeico (CAPE), um componente abundante do própolis de abelhas, com atividade anti-inflamatória, antiviral, antioxidante e imunomodulatória. Estudos têm sugerido seus efeitos benéficos contra as doenças neurodegenerativas, incluindo a doença de Parkinson, e alguns mecanismos têm sido propostos; porém muitos dos estudos com CAPE foram feitos apenas em culturas celulares. Este é o primeiro estudo a demonstrar que a administração intraperitoneal do CAPE protege contra a perda neuronal dopaminérgica e a disfunção motora induzidas pela neurotoxina 6-OHDA em ratos, confirmando a capacidade do CAPE de atravessar a barreira hematoencefálica e exercer seus efeitos benéficos no sitema nervoso central (SNC). Adicionalmente foram empregados dois modelos in vitro para o delineamento de possíveis mecanismos de neuroproteção: (i) mitocôndrias isoladas de cérebro de ratos não tratados e (ii) células SH-SY5Y tratadas com 6-OHDA. Os achados in vivo e in vitro sugerem o envolvimento dos seguintes mecanismos: (i) atividade antioxidante (sequestro de ERO, neutralização de radicais livres e quelação de metais); (ii) atividade anti-inflamatória (inibição da ativação do NF-kB, TNF-?, IkK? e Ikk?); (iii) aumento da expressão da conexina 43; (iv) inibição da Transição de Permeabilidade Mitocondrial (TPM); (v) inibição da liberação de citocromo c e (vi) inibição da ativação da caspase-3, executora final da apoptose. Além disso, o estudo também demonstrou que, por si só, o CAPE não interfere nas funções mitocondriais, o que representa uma vantagem com relação a outros inibidores da TPM. Assim, de acordo com nossos achados, o CAPE é um agente neuroprotetor promissor e pode auxiliar em futuras estratégias terapêuticas para as doenças neurodegenerativas, bem como para o melhor entendimento dos mecanismos responsáveis pelo desenvolvimento dessas doenças. / Parkinson\'s disease (PD) is characterized by progressive loss of dopaminergic neurons in the substantia nigra, which causes various motor dysfunctions. There are no treatments able to delay or stop the degeneration of dopaminergic neurons and the therapy employed nowadays only alleviate the symptoms without altering the progression of PD. In the present study we evaluated the neuroprotective activity of caffeic acid phenethyl ester (CAPE), a compound abundant in honeybees\' propolis, with anti-inflammatory, antiviral, antioxidant and immunomodulatory activities. Its beneficial effects against neurodegenerative diseases, including Parkinson\'s disease, have been suggested and some mechanisms have been proposed; however, many of the studies with CAPE have been performed only in cell cultures. This is the first study to demonstrate that the intraperitoneal administration of CAPE protects against the dopaminergic neuronal loss and the motor dysfunction induced by the neurotoxin 6-OHDA in rats, confirming the ability of CAPE to cross the blood brain barrier and exert its beneficial effects on the central nervous system (CNS). Additionally two in vitro models were used to delineate the possible mechanisms of neuroprotection: (i) mitochondria isolated from the brain of non-treated rats and (ii) cells SH-SY5Y treated with 6-OHDA. The in vivo and in vitro findings suggest the involvement of the following mechanisms: (i) antioxidant activity (scavenger of ROS, free radicals neutralization and metal chelation); (ii) anti-inflammatory activity (inhibition of activation of NF-kB, TNF-?, IkK? and Ikk?); (iii) increased expression of connexin-43; (iv) Inhibition of Mitochondrial Permeability Transition (MPT); (v) inhibition of cytochrome c release (vi) inhibition of the activation of caspase-3, the final executioner of apoptosis. Furthermore, the study also showed that CAPE alone does not interfere with mitochondrial functions, which represents an advantage in relation to other inhibitors of MPT. Therefore, according to our findings, CAPE is a promising neuroprotective agent and may be useful for future therapeutic strategies for neurodegenerative diseases, as well as to better understand the mechanisms responsible for the development of these diseases.

No description available.

A recent model of Parkinson’s disease (PD) suggests that the neuropathological, behavioural and cognitive symptoms progress in stages. There is substantial evidence for a prodromal stage of PD, during which time pre-motor symptoms develop. Rapid eye movement (REM) sleep behaviour disorder (RBD) is a risk factor for developing PD and may be part of the pre-motor stage. In both disorders, neuropathological α-synuclein aggregates are thought to be a direct cause of the resulting symptoms. One model has shown that in rats, proteasome inhibition produced by systemic exposure to environmental toxins results in α-synuclein pathology and motor behaviour dysfunction that mimics the progression of PD in humans. The present study examined the hypothesis that the systemic proteasome inhibition model would produce pre-Parkinsonian RBD-like pathology in rats. It was expected that sleep disturbances would be seen prior to behavioural disturbances in rats treated systemically with PSI (a proteasome inhibitor). Following baseline sleep recording and training on the inclined beam-traverse task, rats were injected with PSI (a proteasome inhibitor) or ethanol (control), 6 times over 2 wk. Sleep recording over 8 wk and behavioural testing over 16 wk provided no evidence of sleep disturbances or motor dysfunction. Post-mortem immunohistochemical analyses of brain tissue provided no evidence of PSI-associated α-synuclein aggregates in the locus coeruleus, subcoeruleus (dorsal part), or substantia nigra (areas involved in RBD and/or PD). These results did not provide support for RBD as a prodromal phase of PD within the systemic proteasome inhibitor-based model and add to a growing body of research reporting inconsistent findings using this model. We suggest that systemic PSI exposure in rats does not produce a viable model of RBD or PD. Whether RBD is an early symptom in the progression of PD remains to be established. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-04-28 12:04:50.613

La disconnessione frontostriatale è considerata il correlato neurale della disfunzione esecutiva nei pazienti con malattia di Parkinson (MP), una patologia neurodegenerativa che si presenta nella fase iniziale come una sindrome clinica asimmetrica con sintomi predominanti nel lato destro o sinistro associati alla perdita neuronale nella sostanza grigia profonda dell'emisfero cerebrale controlaterale. Nella tesi, è stato indagato un particolare aspetto della disfunzione esecutiva nella MP espressa quando è richiesto di selezionare le parole nel contesto di alternative in competizione, cercando differenze in relazione all'insorgenza clinica dei sintomi motori. A livello neurostrutturale, sono state esplorate le alterazioni della sostanza grigia e bianca nelle sottopopolazioni di MP, verificando se la capacità di selezionare le parole è associata ad indici di integrità della materia sostanza grigia e bianca nelle sottopopolazioni di PD. Infine, a livello neurofunzionale, è stato esplorato se la connettività delle regioni cerebrali associate alla disfunzione esecutiva in MP è modulata dalla riserva cognitiva, un fattore di efficienza cognitiva dovuto ad esperienze di vita cognitivamente stimolanti che possono contribuire alla resilienza nel fronteggiare al meglio la neurodegenerazione. Le evidenze raccolte indicano che la lateralità di insorgenza dei sintomi motori e la riserva cognitiva dovrebbero essere prese in considerazione nella valutazione delle funzioni cognitive nei pazienti con MP. / Frontostriatal disconnection is considered the neural correlate of executive dysfunction in patients with Parkinson’s disease (PD), a neurodegenerative pathology that exhibits at the initial phase as an asymmetric clinical syndrome with right or left sided predominant symptoms associated with neuronal loss in the deep grey matter of the contralateral cerebral hemisphere. In the current thesis, it was investigated a particular aspect of the executive dysfunction in PD expressed when it is required to select words in the context of competing alternatives, looking for differences according to the clinical onset of motor symptoms. At the neurostructural level, grey and white matter alterations were explored in PD subpopulations, testing if the ability to select words is associated with indices of grey and white matter integrity in PD subpopulations. Finally, at the neurofunctional level, it was explored if the connectivity of brain regions associated with the executive dysfunction in PD is modulated by the cognitive reserve, a factor of cognitive efficiency due to lifelong experiences that may contribute to the resilience against neurodegeneration. The evidence collected indicates that laterality of clinical motor symptom onset and cognitive reserve should be taken into account in the assessment of cognitive functions in patients with PD.