BACKGROUND: Parkinson’s disease (PD) is a common debilitating disorder that largely effects the aging population. It is associated with a loss of dopamine-producing brain cells, which leads to abnormal brain activity and ultimately, a loss of locomotor control. Transcranial direct current stimulation (tDCS) is a technology that effectively modulates brain excitability by sending low electric current through the scalp. It has been demonstrated to improve working memory, intelligence, learning ability, as well as relieving symptoms of depression, Alzheimer’s and schizophrenia (Kekic, Boysen, Campbell, & Schmidt, 2015; Khedr et al., 2014; Manor et al., 2015). tDCS may thus serve as an effective therapeutic strategy for this vulnerable PD population. OBJECTIVE: The primary purpose of this study was to examine the acute effects of single sessions of tDCS targeting different brain networks on locomotor control metrics and other outcomes in patients with PD. DESIGN: A pilot, double-blinded, sham-controlled study. METHODS: A total of 15 older adults between the ages of 40-85 with a physician diagnosis of PD will be recruited. Participants are screened with questionnaires to determine eligibility. If eligible, participants will undergo a dual task assessment and a freezing of gait (FOG) provoking protocol prior to, as well as immediately after, a 20-minute session of tDCS. The acute effects of each stimulation session will be observed. There will be three different stimulation conditions that each target different areas of the brain: the motor cortex (M1), the motor cortex and the dorsolateral prefrontal cortex (DLPFC), and a sham (i.e., control) condition. Multiple aspects of locomotion (i.e., FOG, gait speed, stride time variability, percent of each walking stride spent with both feet on the ground) and cognition are assessed. RESULTS: This study began enrolling participants on March 3rd, 2016. To date, one participant has been enrolled and completed baseline testing as well as all three tDCS visits. This 42-year-old participant was diagnosed with PD two years ago and symptoms are mild. No side effects were observed during tDCS and the participant was unable to decipher between the M1 and the sham stimulation, but was able to tell the difference between sessions when receiving multi-focal stimulation. DISCUSSION: In this case study, tDCS was well tolerated by the patient and double-blinding procedures were effective. Thus, while tDCS did not induce significant improvements in gait or cognition in this relatively high functioning patient, the developed study protocol and tDCS intervention are highly feasible in the PD population.

Among all of the devastating effects that Parkinson’s disease (PD) has on an individual, sleep dysfunction is one that can have a profound effect on the entire family of the patient. The most potentially destructive of these sleep syndromes being that of Rapid Eye Movement Sleep Behavior Disorder (RBD). This disorder not only causes sleep impairment to the patient, but can occasionally result in life-threatening injury to the individual or their bed partner. While this condition is manageable with medication, the current treatment of choice is a long-acting benzodiazepine, clonazepam. This drug, while effective in treating RBD, comes with a significant burden of side effects. Patients with neurodegenerative disorders, like PD, are at even higher risk of suffering the negative impacts of this treatment. One potential alternative treatment that has been considered is a supplement of exogenous melatonin, a hormone that plays a role in maintaining one’s circadian rhythm. Several small case studies have shown potential efficacy of this treatment, and with very few side effects. However, this efficacy has not yet been proven by randomized clinical trial. This proposed study will perform a double-blind randomized clinical trial of melatonin vs. placebo in a population of PD patients with RBD. Subjects will be analyzed via polysomnographic sleep study, where symptoms will be scored on the RBD Severity Scale (RBDSS) at baseline and after a treatment intervention. Statistical analysis will then ascertain whether or not a significant symptom reduction is seen following melatonin treatment, compared to a group receiving placebo. If melatonin proves to be efficacious in this patient population, this would give clinicians a new treatment option to consider to effectively manage symptoms of RBD with a much lower risk of potentially harmful side effects. Finding an effective method of managing this condition, the prevalence of which continues to rise worldwide, will have a great impact on improving the safety and quality of life of these patients.

Parkinson’s disease (PD) and autism are prevalent diseases in two disparate age groups. The neuropathology underlying these diseases involves the major neurotransmitters, dopamine and GABA, and/ or their receptors. The current study investigated mRNA gene expressions of the GAD67 in autistic striatum and the DRD1 in the Parkinsonian dorsolateral prefrontal cortex. In situ hybridization histochemistry for GAD67 mRNA levels in postmortem striatal specimens from autistic individuals was compared to those of normal controls. Similarly, a nonradioactive in situ hybridization newly emerging method, RNAscope, was used to assess the D1 receptor mRNA gene expression in postmortem specimens of the dorsolateral prefrontal cortex of PD and control brains. The GAD67 mRNA labeling intensity that was measured on X-ray films and on emulsion radioautograph sections did not vary significantly between the autistic samples and the normal control samples. On the other hand, DRD1 mRNA levels showed a significant increase in the Parkinsonian dorsolateral prefrontal cortex specimens as compared to their normal counterparts. The GAD65 mRNA labeling results corresponded with the GAD67 mRNA levels. The similar GAD67 and GAD65 mRNA patterns in the autism group and the control group may suggest that the hyper-excitability hypothesis can be accounted for by an increase in the glutamatergic activity rather than a decrease in the GABAergic system. The increase in the DRD1 mRNA in the Parkinson’s disease dorsolateral prefrontal cortex may be interpreted in light of the expected upregulation of the D1 receptor in cases of dopamine depletion as the treatment-status was unknown. In conclusion, research investigating the neurotransmitters’ gene expression in Parkinson’s disease and in autism spectrum disorder needs more neurobiological studies in order to establish some knowledge regarding the temporality, and the genetic profile mapping of the diseases. Likewise, more research is encouraged to relate the symptoms and behaviors associated with disease to their anatomical origins.

Accumulating evidence links oligomeric species of the protein alpha-synuclein to the neuronal death associated with Parkinson's disease. However, the direct detection of alpha-synuclein oligomers in post-mortem brain has been challenging and this has limited our understanding of their structure, distribution and effects in Parkinson's disease. The work presented in this thesis addresses two aspects of the role of alpha-synuclein oligomers in Parkinson's disease. Firstly, I describe the development of a novel technique, the alpha-synuclein proximity ligation assay (AS-PLA), which specifically detected alpha-synuclein oligomers in vitro and in post-mortem brain tissue. In a blinded study with post-mortem brain tissue from eight Parkinson's disease patients and eight controls, AS-PLA revealed widespread, previously unrecognised pathology in the form of extensive diffuse deposition of alpha-synuclein oligomers. Furthermore, AS-PLA preferentially detected early-stage, loosely compacted Parkinson's disease lesions such as pale bodies, whereas Lewy bodies, considered heavily compacted late lesions were only very exceptionally stained. The oligomeric species detected by AS-PLA displayed a unique, intermediate proteinase K resistance profile, suggesting the detection of a conformer that is different from both physiological pre-synaptic alpha-synuclein (proteinase K sensitive) and highly aggregated alpha-synuclein within Lewy bodies (proteinase K resistant). In addition, AS-PLA revealed the age-dependent accumulation of alpha-synuclein oligomers in the substantia nigra of a BAC transgenic mouse model of Parkinson's disease that overexpresses human wild-type alpha-synuclein, SNCA-OVX. Secondly, the detection of early pathology in Parkinson's disease brain tissue using AS-PLA suggests that oligomeric species of alpha-synuclein could represent a potential target for therapeutic intervention. Therefore, I undertook a screen to identify compounds that can prevent the formation of alpha-synuclein oligomers in vitro. Using bimolecular fluorescence complementation constructs, I identified nine compounds capable of reducing the fluorescence indicative of the formation of alpha-synuclein oligomers. Two of these compounds showed dose-dependent activity. Future work will confirm the hits in vitro before studying whether Parkinson's-like phenotypes in the SNCA-OVX mice can be ameliorated or reversed by treatment with the compounds.

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Salvador, BA, Brasil / Doença de Parkinson (DP) é caracterizada por uma perda seletiva e profunda dos neurônios dopaminérgicos da substância nigra pars compacta (SNpc) do mesencéfalo, acompanhada pela espoliação de dopamina no corpo estriado. A maioria dos casos de DP apresenta etiologia multifatorial, com a presença de componentes genéticos e ambientais. Embora existam diferentes causas possíveis, a patogênese da desordem parece convergir para mecanismos relacionados à disfunção mitocondrial, estresse oxidativo e mau enovelamento proteico. Um modelo estabelecido na literatura para estudo desta doença, tanto in vitro quanto in vivo é a administração de rotenona, um pesticida derivado de plantas que inibe o complexo I mitocondrial e favorece a geração de espécies reativas de oxigênio (ERO), levando a uma espoliação de glutation reduzido (GSH) através do processo de detoxificação destes compostos eletrofílicos, catalisados por glutation S-transferases (GSTs). Sendo assim, a busca por novas substâncias com atividade neuroprotetora é atualmente o foco de estudos, e metabólitos isolados de plantas podem ser fontes destas moléculas. Dessa forma, o 8-metoxipsoraleno (8-MOP), uma furocumarina, foi testado como um possível agente protetor sobre a citotoxicidade causada pela rotenona em modelos in vitro de gliomas, considerando o papel do glutation neste processo. O estudo adotou uma abordagem que associa técnicas bioquímicas e de biologia celular. Ensaios de viabilidade celular foram realizados em células de glioma murino (C6) e glioblastoma multiforme humano (U251) através da redução do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium (MTT), e visualização por microscopia de contraste de fase. O tipo de morte celular provocada pela rotenona nas células U251 foi realizado por marcação com anexina V e iodeto de propídeo (IP), seguido por quantificação por citometria de fluxo. A determinação do conteúdo de GSH intracelular após tratamento com rotenona e 8-MOP foi visualizado por marcação com monoclorobimano (MCB) nas linhagens C6 e U251. Os resultados demonstraram que a rotenona foi citotóxica em ambas as linhagens, reduzindo a viabilidade e alterando a morfologia celular, enquanto que o 8-MOP não apresentou atividade citotóxica. Contudo, o tratamento com o 8-MOP não foi capaz de proteger as células contra os efeitos deletérios da rotenona. No estudo do tipo de morte celular, a porcentagem de células marcadas com anexina V foi maior nos grupos tratados com rotenona, demonstrando que a morte celular ocorre principalmente por apoptose. A análise com MCB demonstrou que a rotenona espoliou GSH, porém o pré-tratamento com 8-MOP inibiu a espoliação. Embora o 8-MOP não tenha sido bem sucedido na proteção das células, a manutenção do conteúdo de GSH corrobora com estudos prévios que descrevem este composto como um potencial inibidor de GST, uma atividade farmacológica que deve ser testada para confirmar a sua eficácia como agente terapêutico. / Parkinson’s disease (PD) is characterized by a profound and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) accompanied by midbrain dopamine depletion in the striatum. Most cases of PD present multifactorial etiologies, with the presence of genetic and environmental components. Although there are different possible causes, the pathogenesis of the disorder seems to converge to mechanisms related to mitochondrial dysfunction, oxidative stress and bad protein folding. An established model in the literature to study this disease, both in vitro and in vivo is rotenone administration, a pesticide derived from plants that inhibits the mitochondrial complex I and favors the generation of reactive oxygen species (ROS), leading to reduced glutathione (GSH) depletion through the detoxification process of this electrophilic compound catalyzed by glutathione S-transferases (GSTs). Thus, the search for new substances with neuroprotective activity is currently the focus of studies, and plant isolated metabolites can be sources of these molecules. Thus, 8-methoxypsoralen (8-MOP), a furocoumarin, was tested as a potential protective agent on the cytotoxicity caused by rotenone in glioma cells in vitro models, considering the role of glutathione in the process. The study adopted an approach that combines biochemical and cell biology techniques. Cell viability assays were performed in murine glioma cells (C6) and human glioblastoma (U251) cells through the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and visualization by phase contrast microscopy. The type of cell death caused by rotenone in U251 cells was performed by staining with annexin V and propidium iodide (PI) followed by flow cytometric quantitation. The determination of intracellular GSH content after treatment with rotenone and 8-MOP was visualized by staining with monochlorobimane (MCB) in the lineages U251 and C6. The results demonstrated that rotenone was cytotoxic to both cell lineages, reducing the viability and changing the cell morphology, whereas the 8-MOP did not show cytotoxic activity. However, the treatment with 8-MOP was not able to protect cells against the deleterious effects of rotenone. In the type of cell death studies, the percentage of cells stained with annexin V was higher in the groups treated with rotenone, demonstrating that cell death occurs primarily by apoptosis. The analysis with MCB has shown that rotenone depleted GSH, but pre-treatment with 8-MOP inhibited the depletion. Although the 8-MOP has not been successful in protecting cells, the maintenance of GSH content corroborates with previous studies that describe this compound as a potential inhibitor of GST, a pharmacological activity that should be tested to confirm its effectiveness as a therapeutic agent.

Ubiquitin phosphorylation by PINK1 (PTEN-induced Putative Kinase 1) is crucial for mitochondrial quality control and loss or mutation of PINK1 can lead to autosomal recessive juvenile parkinsonism (AR-JP). PINK1 is an unusual kinase, as it is characterised by three unique insertions in its kinase N lobe and a C-terminal region after the kinase domain. Despite great effort, a structure of PINK1 could not be determined and the molecular mechanism of ubiquitin phosphorylation and the effect of the PINK1 AR-JP patient mutations remained elusive. The versatile modifier ubiquitin (Ub) is also an unusual kinase substrate, as its phosphorylation site (Ser65) is not exposed, but protected by the Ub fold. Hence, it was not clear how a kinase would be able to target Ser65 of Ub. This work shows that Ub needs to adopt a previously described conformation in order to be efficiently phosphorylated by PINK1. NMR experiments revealed that in a small population of Ub the last β-strand is retracted, resulting in a more accessible Ser65 loop. It could be shown that PINK1 binds the Ser65 loop in this C-terminally retracted conformation (Ub-CR), but not in the ‘common’ conformation. In addition, it could be shown that Ub trapped in the Ub-CR conformation by point mutations (Ub TVLN) is phosphorylated significantly faster than Ub wt, which only adopts the Ub-CR conformation at very low frequency. To further elucidate how PINK1 binds and phosphorylates Ub, the kinase domain of Pediculus humanus corporis (Ph)PINK1 was crystallised in complex with Ub TVLN stabilised by a nanobody. The structure revealed many peculiarities of PINK1, such as the architecture of the unique insertions and the C-terminal region. Together with NMR and mass spectrometry studies, the structure explains how PINK1 interacts with ubiquitin via insertion-3 and its activation segment, and how PINK1 utilises the Ub- CR conformation for efficient Ser65 phosphorylation. In addition, the structure shows that two autophosphorylation sites in the N lobe regulate PINK1, by stabilising the functionally important insertions. The structure helped our understanding of the molecular basis of over 40 AR-JP patient mutations and may guide the design of ARJP therapeutics in the future.

This study investigated problem solving performance in Parkinson's disease (PD) individuals, PD individuals in collaboration with their caregiving spouses, as well as in healthy older adult individuals and collaborating couples. Problem solving abilities represent executive functions mediated by frontal cortex. Given frontal lobe involvement in PD, the supporting neuropsychological evidence indicates problem solving deficits in this patient population. The extent to which these individual-level deficits could be overcome (or compensated) through collaboration was explored. Two groups of elderly married couples participated in the study. The control group consisted of 20 healthy couples with neither partner having a medical diagnosis of PD. The experimental group comprised 17 couples in which the male spouse had received a diagnosis of PD from a qualified neurologist. All participants met several selection criteria: (a) aged 55 years or older, (b) relatively well-educated for their age cohort, (c) above a criterion in mental status, and (d) below a clinical criteria of depression. They performed three problem solving tasks: verbal fluency, the Wisconsin Card Sorting Test (WCST), and the 20 Questions task. These tasks were performed twice—once individually and once collaboratively with their spouse. This within-subjects group size variable was counterbalanced so that half of the subjects were tested first as individuals and then as dyads and vice versa. The collaborative part of each testing session was videotaped. The results indicated: (a) poorer performance by the experimental couples and Parkinsonian men relative to the other participants on qualitative indices of the verbal fluency task, (b) a detrimental effect of collaboration on the speeded verbal fluency task (c) group level benefit of collaboration and inferred individual-level benefit to the Parkinsonian men for some measures on the card sorting task, (d) a benefit of collaboration for the experimental group on the 20 Questions task, (e) greater verbal input to the process of solving the 20 Questions task by the experimental females apparently to compensate for their Parkinsonian husbands, and (f) differences between the control and experimental groups in the process variables that were related to efficient questioning strategies on the 20 Questions task. Theoretical and clinical implications of this research are discussed. Limitations and possible directions for future investigation are noted. / Graduate

abstract: Although tremor, rigidity, and bradykinesia are cardinal symptoms of Parkinson's disease (PD), impairments of gait and balance significantly affect quality of life, especially as the disease progresses, and do not respond well to anti-parkinsonism medications. Many studies have shown that people with PD can walk better when appropriate cues are presented but, to the best of our knowledge, the effects of real-time feedback of step length and uprightness of posture on gait and posture have not been specifically investigated. If it can be demonstrated that real-time feedback can improve posture and gait, the resultant knowledge could be used to design effective rehabilitation strategies to improve quality of life in this population. In this feasibility study, we have developed a treadmill-based experimental paradigm to provide feedback of step length and upright posture in real-time. Ten subjects (mean age 65.9 ± 7.6 years) with mild to moderate PD (Hoehn and Yahr stage III or below) were evaluated in their ability to successfully utilize real-time feedback presented during quiet standing and treadmill walking tasks during a single data collection session in their medication-on state. During quiet standing tasks in which back angle feedback was provided, subjects were asked to utilize the feedback to maintain upright posture. During treadmill walking tasks, subjects walked at their self-selected speed for five minutes without feedback, with feedback of back angle, or with feedback of step length. During walking tasks with back angle feedback, subjects were asked to utilize the feedback to maintain upright posture. During walking tasks with step length feedback, subjects were asked to utilize the feedback to walk with increased step length. During quiet standing tasks, measurements of back angle were obtained; during walking tasks, measurements of back angle, step length, and step time were obtained. Subjects stood and walked with significantly increased upright posture during the tasks with real-time back angle feedback compared to tasks without feedback. Similarly, subjects walked with significantly increased step length during tasks with real-time step length feedback compared to tasks without feedback. These results demonstrate that people with PD can utilize real-time feedback to improve upright posture and gait. / Dissertation/Thesis / Masters Thesis Bioengineering 2014

abstract: Parkinson's disease (PD) is a neurodegenerative disorder that produces a characteristic set of neuromotor deficits that sometimes includes reduced amplitude and velocity of movement. Several studies have shown that people with PD improved their motor performance when presented with external cues. Other work has demonstrated that high velocity and large amplitude exercises can increase the amplitude and velocity of movement in simple carryover tasks in the upper and lower extremities. Although the cause for these effects is not known, improvements due to cueing suggest that part of the neuromotor deficit in PD is in the integration of sensory feedback to produce motor commands. Previous studies have documented some somatosensory deficits, but only limited information is available regarding the nature and magnitude of sensorimotor deficits in the shoulder of people with PD. The goals of this research were to characterize the sensorimotor impairment in the shoulder joint of people with PD and to investigate the use of visual feedback and large amplitude/high velocity exercises to target PD-related motor deficits. Two systems were designed and developed to use visual feedback to assess the ability of participants to accurately adjust limb placement or limb movement velocity and to encourage improvements in performance of these tasks. Each system was tested on participants with PD, age-matched control subjects and young control subjects to characterize and compare limb placement and velocity control capabilities. Results demonstrated that participants with PD were less accurate at placing their limbs than age-matched or young control subjects, but that their performance improved over the course of the test session such that by the end, the participants with PD performed as well as controls. For the limb velocity feedback task, participants with PD and age-matched control subjects were less accurate than young control subjects, but at the end of the session, participants with PD and age-matched control subjects were as accurate as the young control subjects. This study demonstrates that people with PD were able to improve their movement patterns based on visual feedback of performance and suggests that this feedback paradigm may be useful in exercise programs for people with PD. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2015

abstract: Progressive gait disorder in Parkinson's disease (PD) is usually exhibited as reduced step/stride length and gait speed. People with PD also exhibit stooped posture, which can contribute to reduced step length and arm swing. Since gait and posture deficits in people with PD do not respond well to pharmaceutical and surgical treatments, novel rehabilitative therapies to alleviate these impairments are necessary. Many studies have confirmed that people with PD can improve their walking patterns when external cues are presented. Only a few studies have provided explicit real-time feedback on performance, but they did not report how well people with PD can follow the cues on a step-by-step basis. In a single-session study using a novel-treadmill based paradigm, our group had previously demonstrated that people with PD could follow step-length and back angle feedback and improve their gait and posture during treadmill walking. This study investigated whether a long-term (6-week, 3 sessions/week) real-time feedback training (RTFT) program can improve overground gait, upright posture, balance, and quality of life. Three subjects (mean age 70 ± 2 years) with mild to moderate PD (Hoehn and Yahr stage III or below) were enrolled and participated in the program. The RTFT sessions involved walking on a treadmill while following visual feedback of step length and posture (one at any given time) displayed on a monitor placed in front of the subject at eye-level. The target step length was set between 110-120% of the step length obtained during a baseline non-feedback walking trial and the target back angle was set at the maximum upright posture exhibited during a quiet standing task. Two subjects were found to significantly improve their posture and overground walking at post-training and these changes were retained six weeks after RTFT (follow-up) and the third subject improved his upright posture and gait rhythmicity. Furthermore, the magnitude of the improvements observed in these subjects was greater than the improvements observed in reports on other neuromotor interventions. These results provide preliminary evidence that real-time feedback training can be used as an effective rehabilitative strategy to improve gait and upright posture in people with PD. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2017