<p>Parkinson’s disease (PD) is a complex neurodegenerative movement disorder involving protein misfolding, mitochondrial dysfunction, and oxidative stress. The current dissertation, motivated by a lack of valid biomarkers and sustainable therapies, examined the potential application of a novel target for therapeutics and diagnostics of PD — the multifunctional, heat-shock like protein Catecholamine-Regulated Protein 40 (CRP40). The goal of this program of research was to elucidate further the implications of CRP40 in PD using a variety of molecular biology, bioinformatics, and clinical approaches through integrative collaborations with academia, government, and industry partners to translate scientific findings into real world solutions. Chapters 2 and 3 explored the potential therapeutic use and structure-function relationships of CRP40 through elucidating the smallest functional piece of this protein that was six times smaller, and validating a negative control for these experiments (Heat-Shock Protein 47). These initiatives could eventually lead to a small drug that could cross the blood-brain barrier and be targeted to the specific brain regions affected in PD. Chapter 4 examined the potential mechanisms of CRP40, and suggested that this protein may protect neurons from oxidative stress, maintain energy levels, and mitochondrial homeostasis, with important future implications for a variety of disorders. Finally, Chapter 5 presented compelling evidence for the potential use of CRP40 as a valid biomarker for early detection of PD and monitoring of disease progression. Overall, findings suggest that CRP40 may be a critical target for future breakthroughs in the diagnosis and treatment of PD.</p> / Doctor of Science (PhD)

Neuronal activity in the brain is tightly regulated. During operation in real time, for instance, feedback and feedforward loops limit excessive excitation. In addition, cell autonomous processes ensure that neurons’ average activity is restored to a setpoint in response to chronic perturbations. These processes are summarized as homeostatic plasticity (Turrigiano in Cold Spring Harb Perspect Biol 4:a005736–a005736, 2012). In the basal ganglia, information is mainly transmitted through disinhibition, which already constraints the possible range of neuronal activity. When this tightly adjusted system is challenged by the chronic decline in dopaminergic neurotransmission in Parkinson’s disease (PD), homeostatic plasticity aims to compensate for this perturbation. We here summarize recent experimental work from animals demonstrating that striatal projection neurons adapt excitability and morphology in response to chronic dopamine depletion and substitution. We relate these cellular processes to clinical observations in patients with PD that cannot be explained by the classical model of basal ganglia function. These include the long duration response to dopaminergic medication that takes weeks to develop and days to wear off. Moreover, dyskinesias are considered signs of excessive dopaminergic neurotransmission in Parkinson’s disease, but they are typically more severe on the body side that is more strongly affected by dopamine depletion. We hypothesize that these clinical observations can be explained by homeostatic plasticity in the basal ganglia, suggesting that plastic changes in response to chronic dopamine depletion and substitution need to be incorporated into models of basal ganglia function. In addition, better understanding the molecular mechanism of homeostatic plasticity might offer new treatment options to avoid motor complications in patients with PD.

Abstract: Background: In Parkinson’s disease, postural instability and falls are of particular socioeconomic relevance. Although effective fall prevention and the prophylaxis of fall-related injuries depend on low-threshold symptom monitoring, validated instruments are lacking. Objectives: To develop a self-report questionnaire for the assessment of falls, near falls, fear of falling, fallrelated injuries, and causes of falls for patients with Parkinson’s disease (PwPD). - Methods: A pool of potential items was generated from a literature review and by discussion in an expert panel. The first version of the Dresden Fall Questionnaire (DREFAQ) was tested in a group of German-speaking movement disorder specialists as well as PwPD. The resulting 5-item questionnaire was assessed in a validation cohort of 36 PwPD who documented fall events and near-fall events in a calendar for 3 months and completed the DREFAQ at the end of the study. The questionnaire was subsequently used in a separate cohort of 46 PwPD to determine test–retest reliability and confirm the factor structure. - Results: The DREFAQ showed good internal consistency (Cronbach’s α = 0.84) and good test–retest reliability (intraclass correlation coefficient, 0.76; 95% confidence interval, 0.60–0.86). The total DREFAQ score showed good concurrent validity with fall events (Spearman’s ρ = 0.82) and near-fall events (Spearman’s ρ = 0.78) as determined by fall and near-fall diaries. Factor analysis revealed a 2-factor structure composed of near falls with fear of falling and severe falls with injuries. - Conclusions: The DREFAQ is a reliable and valid 5-item questionnaire for determining the incidence of falls, near falls, fear of falling, fall-related injuries, and causes of falls in PwPD.

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aβ(1-42) and pGlu-Aβ(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aβ(1-42) and pGlu-Aβ(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aβ in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aβ and pGlu-Aβ, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aβ species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

In Parkinson's disease, aggregates of α-synuclein within Lewy bodies and Lewy neurites represent neuropathological hallmarks. However, the cellular and molecular mechanisms triggering oligomeric and fibrillary α-synuclein aggregation are not fully understood. Recent evidence indicates that oxidative stress induced by metal ions and post-translational modifications such as phosphorylation, ubiquitination, nitration, glycation, and SUMOylation affect α-synuclein conformation along with its aggregation propensity and neurotoxic profiles. In addition, proteolytic cleavage of α-synuclein by specific proteases results in the formation of a broad spectrum of fragments with consecutively altered and not fully understood physiological and/or pathological properties. In the present review, we summarize the current knowledge on proteolytical α-synuclein cleavage by neurosin, calpain-1, cathepsin D, and matrix metalloproteinase-3 in health and disease. We also shed light on the contribution of the same enzymes to proteolytical processing of pathogenic proteins in Alzheimer's disease and report potential cross-disease mechanisms of pathogenic protein aggregation.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79- α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may—in analogy to pGlu-Aβ peptides in Alzheimer’s disease—act as a seed for pathogenic protein aggregation.

Introduction: The OPTIPARK study confirmed the effectiveness and safety of opicapone as adjunct therapy to levodopa in patients with Parkinson’s disease (PD) and motor fluctuations under real-world conditions. The aim of this sub-analysis was to evaluate opicapone in the German patient cohort of OPTIPARK in order to provide country-specific data. Methods: OPTIPARK was an open-label, single-arm study conducted in routine clinical practice across Germany and the UK. Patients with PD and motor fluctuations received once-daily opicapone 50 mg for 3 months in addition to levodopa. The primary endpoint was Clinicians’ Global Impression of Change (CGI-C). Secondary assessments included Patients’ Global Impressions of Change (PGI-C), Unified Parkinson’s Disease Rating Scale (UPDRS) I–IV, Parkinson’s Disease Questionnaire (PDQ-8), and Non-Motor Symptoms Scale (NMSS). This sub-analysis reports outcomes from the German patients only. Results: Overall, 363 (97.6%) of the 372 patients included in the German cohort received ≥1 dose of opicapone and 291 (80.2%) completed the study. Improvements on CGI-C and PGI-C were reported by 70.8% and 76.3% of patients, respectively. UPDRS scores improved for activities of daily living during OFF time by −3.3 ± 4.5 points and motor scores during ON time by −5.3 ± 7.9 points. PDQ-8 and NMSS scores also demonstrated improvements. Treatment emergent adverse events considered at least possibly related to opicapone occurred in 37.7% of patients, with most being of mild or moderate intensity. Conclusion: Opicapone added to levodopa in patients with PD and motor fluctuations was effective and generally well tolerated in routine clinical practice across Germany.

The question whether life style may impair the advent or course of the disease in patients with Parkinsonism is of great importance for patients and physicians alike. We present here comprehensive information on the influence of the environment, diet (especially caffeine, nicotine, alcohol, chocolate and dairy products), physical activity and sleep on risk and course of Parkinson’s disease.

Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015

Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015