<div><p>Parkinson’s disease (PD) is a devastating neurodegenerative disorder that affects 10 million people worldwide and is characterized by pronounced motor symptoms. Dementia with Lewy Bodies (DLB) involves both cognitive and motor deficits and affects ~1 million people in the United States. To date there is no cure for PD or DLB, and current treatments address only a subset of the symptoms that define these diseases. PD and DLB are ‘synucleinopathies’, defined as disorders involving the accumulation in patients’ brains of Lewy bodies. Lewy bodies are cellular inclusions that consist largely of aggregated species of alpha-synuclein (aSyn), a presynaptic protein that exists as both cytosolic and membrane-bound forms. Pathophysiological findings suggest that aggregated aSyn is involved in neurodegeneration in PD and DLB. However, mechanisms by which aSyn forms neurotoxic aggregates, and neurotoxic processes that distinguish different synucleinopathies such as PD and DLB, are poorly understood. To address these gaps, we have (i) designed a protocol to establish a primary cell culture model that can recapitulate key neuropathological features of PD, (ii) examined effects of expressing aSyn variants in a rat model of PD, and (iii) examined the expression profiles of neuroprotective genes in PD and DLB brain specimens.</p><p> </p><p>In the first part of my thesis, I describe the development of an optimized protocol to prepare primary midbrain and cortical cultures from rat embryonic brains for the study of PD and other synucleinopathies. The establishment of cellular models that simulate specific aspects of neuropathology can enable the characterization of molecular perturbations that lead to dopaminergic (DA) neuronal death. Our primary midbrain mixed culture model provides an outstanding opportunity to explore therapeutic strategies to rescue DA neurons from toxicity elicited by a range of PD-related insults. In addition, our primary cortical mixed cultures can be used to model cortical neuropathology in various CNS disorders including synucleinopathies.</p><p> </p><p>A number of mutations in the gene that codes for aSyn are associated with familial, early-onset forms of PD. A major goal of my thesis research is to characterize neurotoxic effects of a recently discovered familial substitution, A53E. This mutant was chosen based on the rationale that the introduction of a negatively charged residue at position 53 could potentially interfere with aSyn-membrane interactions and favor A53E aggregation, as we described for other familial aSyn mutants. For the first time, we have reproduced the neurotoxicity of A53E seen in human patients by expressing the mutant protein in rat midbrain. Rats injected unilaterally in the substantia nigra (SN) with rAAV encoding A53E and another familial mutant, A53T, but not rAAV encoding WT aSyn or a vector-control (‘stuffer’) virus, exhibited a significant motor impairment. Immunohistochemical analysis at 14 weeks after the viral injection revealed that brain sections from aSyn-expressing rats exhibit key features reminiscent of neuropathology in human PD, including nigral dopaminergic neuron loss (confirmed by unbiased stereology), striatal terminal depletion, and aSyn inclusion formation. In addition, it was determined that WT aSyn and the A53E and A53T mutants invaded the non-injected substantia nigra, implying that expressed aSyn protein can spread throughout the brain in the rat rAAV-aSyn model. These results yield insights into the molecular basis for the neurotoxicity of A53E and shed light on a potential role for membrane-induced aSyn aggregation in PD pathogenesis in vivo, thus setting the stage for developing therapies to slow neurodegeneration in the brains of familial and idiopathic PD patients. </p><p> </p><p>aSyn neurotoxicity varies with the expression of neuroprotective proteins, and misfolded aSyn affects cellular functions and gene expression. These observations suggest that differential gene expression patterns can inform us about similarities and differences in pathogenic mechanisms of different synucleinopathy disorders. A third phase of my thesis research was aimed at determining the expression levels of a panel of candidate neuroprotective genes in post-mortem brain samples from DLB and PD patients and age-matched controls (5 individuals in each group). mRNAs encoding the following proteins were quantified via qRT-PCR in homogenates prepared from the frontal cortex and the BA24 region encompassing the cingulate gyrus: DJ-1, a protein with antioxidant and chaperone activities; PGC1α, a master regulator of mitochondrial biogenesis and oxidative metabolism; MsrA, an antioxidant enzyme responsible for repairing oxidatively damaged proteins; and ATP13A2, a lysosomal protein involved in autophagy. In addition to yielding new insights into differential gene expression patterns in cortex versus cingulate gyrus, the data revealed differences in mRNA expression levels in DLB versus non-DLB cortical tissue. Although levels of all four neuroprotective mRNAs were increased (or showed a trend towards being increased) in DLB cortex, Western blot analysis revealed that only the DJ-1 and PGC1α proteins showed a trend towards being up-regulated, whereas levels of ATP13A2 and MsrA were unchanged. These findings suggest that there is a failure to induce cellular antioxidant responses and lysosomal autophagy at the protein level in DLB cortex, and in turn this failure could contribute to neuropathology. Interestingly, analysis of the same panel of neuroprotective genes in PD cortical samples did not show significant differences in mRNA or protein levels compared to control samples, suggesting that different neuroprotective mechanisms are induced in DLB versus PD cortex. These studies shed light on brain-region specific changes in gene expression associated with different synucleinopathy disorders, and they set the stage for developing new diagnostic tests and therapeutic strategies.</p></div><br>

No description available.

Ein Schlüsselbefund der Parkinson-Krankheit auf zellulärer Ebene ist das Auftreten von Protein-Einschlusskörperchen, sogenannten Lewykörperchen. Der Hauptbestandteil dieser Lewykörperchen ist pathologisch aggregiertes, fibrilläres α-Synuklein, ein Protein, welches Einfluss auf präsynaptische Vesikel, Protein- und Enzymfunktionen sowie den Dopaminstoffwechsel und den axonalen Transport hat. Bis heute ungeklärt ist die Ursache der Aggregation des Proteins. Zahlreiche Forschungsaktivitäten werden in diese Richtung unternommen. Die pathologischen Mechanismen, die zur abnormen Aggregation von α-Synuklein führen, bleiben noch weitgehend unbekannt. Ein großer Teil der Literatur unterstützt die Hypothese, dass α-Synuklein bei Punktmutationen oder erhöhter Expression anfällig für Aggregationen ist und damit Neuronen geschädigt werden. Die Einzelheiten dieses sukzessiven Aggregationsprozesses und die Mechanismen, die dabei letztlich den Zelltod verursachen, bleiben unklar. Alterungsprozesse und Umweltfaktoren sind entscheidende Risikofaktoren. Obwohl es immer mehr Hinweise gibt, dass α-Synuklein-Aggregate eine wichtige pathophysiologische Rolle spielen, wird bisher noch unzulänglich verstanden, wie die für die dopaminergen Neurone toxische Wirkung entfaltet wird. Als gesicherter Pathomechanismus der Degeneration der dopaminergen Nervenzellen gilt ein erhöhter oxidativer Stress. Es wird vermutet, dass er zur Aggregation des α-Synukleins beitragen kann. Ziel dieser vorgelegten Arbeit ist es, durch die Verwendung eines geeigneten Zellkulturmodells zur Aufklärung der beschriebenen pathologischen Mechanismen beizutragen. In dieser Studie wurden zwei artifizielle Modellsubstanzen in einer dopaminergen Primärzellkultur eingesetzt, die Pestizide Rotenon und Paraquat, um die pathologischen Verhältnisse in der Substantia nigra zu simulieren. Sie erzeugen oxidativen Stress durch Hemmung der mitochondrialen Atmungskette bzw. Redoxreaktionen mit molekularem Sauerstoff, was zum dopaminergen Zelltod führt. Im Rahmen dieser Studie gelang es, beide Parkinson-Zellkulturmodelle anhand der Lokalisierung, des Aggregationsverhaltens, des Einflusses auf die Mikroglia-Aktivierung sowie des Abbaus von α-Synuklein näher zu charakterisieren. Hierzu wurde α-Synuklein durch Fluoreszenzfärbung, Westernblot und Immunpräzipitation analysiert. Eine kurzzeitige Behandlung mit hoch konzentrierten Toxinen löst eine akute Degeneration dopaminerger Neurone aus, die so nicht der des Idiopathischen Parkinsons entspricht. Beim IPS erfolgt die Degeneration über Jahre hinweg. Um auch den Einfluss der zellulären Alterung auf die α-Synuklein-Aggregation zu zeigen, wurden die in dieser Arbeit verwendeten Zellkulturen über 46 Tage kultiviert und die Pestizid-Konzentration so eingestellt, dass etwa 25-50 % der dopaminergen Neurone absterben. Es konnte gezeigt werden, dass es nach chronischer Behandlung mit dem jeweiligen Pestizid zu den verschiedenen Zeitpunkten Unterschiede in der Lokalisation sowie in der Konformation von α-Synuklein gibt. Die zum Vergleich nur bis zum Tag 11 kultivierten Zellkulturen zeigten nach kurzer Behandlung mit hochkonzentrierter Toxin-Menge eine Ansammlung von α-Synuklein im Soma, aber keine Auswanderung und Lokalisierung in und an den Neuriten, wie es nach chronischer Rotenon-Behandlung beobachtet werden konnte. Bei den Rotenon-behandelten Zellen ist ein Prozess der Verlagerung und Anhäufung des α-Synuklein aus dem Soma in die Neuriten bereits ab einem früheren Zeitpunkt zu beobachten als in den Kontrollen, welche sich aber mit zunehmendem Alter ähnlich verhalten. Außerdem konnte in den Kulturen, besonders bei den Rotenon-behandelten dopaminergen Neuronen, ein punktförmiges Verteilungsmuster und größere Ansammlungen des Proteins in den Neuriten beobachtet werden, was für eine aggregierte Form des α-Synukleins spricht. Diese Aggregate ließen sich durch die Proteo-Aggreosom-Färbung nachweisen. Auch der Nachweis von am Serin 129 phosphoryliertem α-Synuklein in diesen größeren Ansammlungen gilt als Zeichen für eine aggregierte Form. Die Beobachtung, dass α-Synuklein mit zunehmendem Kulturalter aus dem Soma in die Peripherie austritt, konnte bei der chronischen Paraquat-Behandlung so nicht getätigt werden. Unter Paraquat-Behandlung war keine Herauf-Regulierung der Gesamt-α-Synuklein Expression in der Kultur zu beobachten, wie dies sowohl bei Kontrolle als auch bei Rotenon der Fall ist. Wir vermuten im Paraquat-Modell, dass die sich im Soma ansammelnde Form von α-Synuklein durch vermehrte Einschleusung in den Zellkern zur Toxizität beitragen kann. Auch eine Interaktion von α-Synuklein mit der Zellmembran könnte unter Paraquat-Einfluss zum dopaminergen Zelltod beitragen. Durch Immunpräzipitation und Fluoreszenz-Doppelfärbung von α-Synuklein und Ubiquitin konnten wir den Abbau des fehlgefalteten α-Synukleins in der Zelle durch Ubiquitinierung nachweisen. Unter Rotenon ist ab DIV 14 eine starke Kolokalisation von α-Synuklein und Ubiquitin zu erkennen, die im Verlauf der Kultur nachlässt und nur noch in punktförmigen Aggregaten außerhalb der Zelle zu sehen ist. Unter Paraquat zeigte sich während der gesamten Kultivierung Polyubiquitinierung und Kolokalisation der beiden Proteine in der gesamten Zelle. Die Aggregationsform von α-Synuklein scheint das Proteinabbausystem durch Ubiquitinierung zu beeinflussen, da wir davon ausgehen, dass es sich bei der α-Synuklein-Konformation zu verschiedenen Zeitpunkten in den Paraquat-behandelten dopaminergen Neuronen nicht um die Aggregationsform handelt, die wir unter Rotenon beobachten konnten.

No description available.

Small extracellular vesicles (small EVs) are endogenous membrane-enclosed nanocarriers released from essentially all cells. They have been shown to carry proteins, lipids, nucleic acids to transmit biological signals throughout the body, including to the brain. Some evidence has suggested that small EVs can cross the blood-brain barrier (BBB), moving from the peripheral circulation to the central nervous system (CNS). The BBB is a dynamic barrier that regulates molecular trafficking between the peripheral circulation and the CNS. As a result, small EVs have attracted attention for their potential as a novel delivery platform for nucleic acid-based therapeutics across the BBB. Silencing RNAs (siRNAs) are a potent drug class but using “naked” siRNA is not feasible due to their short half-life, vulnerability to degradation and low penetration in cells. Despite the excitement for the development of small EV-based therapeutics, their clinical development is hampered by the lack of reliable methods for packing therapeutics into them. Reshke et al. has shown that cells can be genetically engineered to produce customizable small EVs packaged with siRNA against any protein by integrating the siRNA sequence into the pre- miR-451 structure. Mounting evidence has established that in a misfolded state, α-synuclein becomes insoluble and phosphorylated to form intracellular inclusions in neurons (known as Lewy bodies) which leads to Parkinson’s disease (PD) pathogenesis. Given that increased α-synuclein expression causes familial and idiopathic PD, decreasing its synthesis by using siRNA is an attractive therapeutic strategy. Here, we genetically engineered cells to produce small EVs packaged with siRNA against α-synuclein integrated in the pre-miR451 backbone, tested their ability to cross an in vitro BBB, and deliver its cargo to silence endogenous α-synuclein in neuron- like cells. The therapeutic potential of α-synuclein siRNA delivery by these small EVs was demonstrated by the strong mRNA (60-70%) and protein knockdown (43%) of α-synuclein in neuron-like cells. We also demonstrated that approximately at 4% and 2%, respectively of small EVs-derived from human brain endothelial cells (hCMEC/D3) and human embryonic kidney (HEK293T) were transported cross the in vitro BBB model. Interestingly, we observed that small EVs-derived from HEK293T deliver their cargo to induced brain endothelial cells (iBECs) (~74% α-synuclein mRNA reduction) but their rate of transport across BBB was lower and did not reduce α-synuclein mRNA expression in neuron-like cells, seeded on the far side of the BBB. Small EVs- derived from hCMEC/D3 reduced α-synuclein mRNA (40%) in neuron-like cells across the BBB model. This finding suggests that small EVs derived from different cell sources can undergo different intracellular trafficking routes, providing various opportunities to influence the efficiency of delivery and fate of intracellular cargo. Using small EVs-derived from hCMEC/D3, two different routes of administration, a single bolus intravenous (IV) or intra-carotid (ICD) injection, showed small EVs largely accumulated in the liver, spleen, small intestines and kidneys; and only a small amount of small EVs were detected in the brain. These results indicate that human brain endothelial cells may serve as a promising cell source for CNS treatments based on small EVs.

No description available.

<p>The proteasome is a multi-subunit protease complex responsible for most of the non-lysosomal protein turnover in eukaryotic cells. This degradation process can be conducted dependent or independent of ubiquitination as different isoforms with different substrate preferences coexist in the cell. Proteasomal activity declines during aging due to a decreased expression of proteasome subunits, complex disassembly, and oxidative stress. This malfunction leads to protein accumulation, subsequent aggregation, and ultimately diseased states. Considering the shared feature of aggregation and accumulation of intrinsically disordered proteins (IDPs) in age-related diseases, and the substrate preference of the 20S isoform for misfolded proteins, enhancing the proteolytic activity of the 20S proteasome has arisen as an attractive strategy to minimize the burden associated with this increased protein load. Recently, we identified the FDA-approved drug miconazole (MO) as a stimulator of the 20S isoform and validated its activity profile in biochemical and cell-based assays. Given its FDA-approved drug status, we considered that to successfully repurpose it, information regarding its binding location within the 20S and network of binding partners, as well as its value in protein homeostasis in age-related diseases are necessary. Herein, we (1) conduct SAR studies to determine MO’s key features responsible for proteasomal activation and obtain molecules with enhanced ability to activate the 20S proteasome; next, using the developed SAR model, we (2) design a diazirine-based photoreactive probe that allows for the identification of MO’s binding partners and location within the 20S proteasome. Lastly, we (3) explore the use of MO to restore the activity of impaired proteasomes by Parkinson’s disease-associated toxic oligomers. This work expands upon previous research avenues by using newer approaches to study this enzymatic complex, and describes methods that can be further used to better establish the role of the 20S proteasome in age-related diseases.</p>

The deposition of β-amyloid peptides and of α-synuclein proteins is a neuropathological hallmark in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) subjects, respectively. However, there is accumulative evidence that both proteins are not exclusive for their clinical entity but instead co-exist and interact with each other. Here, we investigated the presence of a newly identified, pyroglutamate79-modified α-synuclein variant (pGlu79-aSyn)—along with the enzyme matrix metalloproteinase-3 (MMP-3) and glutaminyl cyclase (QC) implicated in its formation—in AD and in the transgenic Tg2576 AD mouse model. In the human brain, pGlu79-aSyn was detected in cortical pyramidal neurons, with more distinct labeling in AD compared to control brain tissue. Using immunohistochemical double and triple labelings and confocal laser scanning microscopy, we demonstrate an association of pGlu79-aSyn, MMP-3 and QC with β-amyloid plaques. In addition, pGlu79-aSyn and QC were present in amyloid plaque-associated reactive astrocytes that were also immunoreactive for the chaperone heat shock protein 27 (HSP27). Our data are consistent for the transgenic mouse model and the human clinical condition. We conclude that pGlu79-aSyn can be generated extracellularly or within reactive astrocytes, accumulates in proximity to β-amyloid plaques and induces an astrocytic protein unfolding mechanism involving HSP27.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide affecting 1 - 2 % of the population older than 65. Patients develop characteristic motoric dysfunctions alongside early-onset non-motor symptoms including sleeping disorders, anxiety or depression and late-stage cognitive deficits such as dementia. To date, dopamine-replacement therapies are the gold standard for treating PD patients, improving motoric disorders by compensating for the loss of dopaminergic neurons in the substantia nigra, however no curative therapies to prevent disease progression are yet available. The pathomechanism underlying PD is complex, and the interplay of factors causing the disease is not entirely understood. The formation of α-synuclein protein aggregates, being one of the hallmarks associated with PD, is regarded as a major contributor to neuronal death and the spreading of PD pathology throughout different brain regions as the disease progresses. In the past, deficits in cellular protein clearance machinery have been affiliated with the accumulation of α-synuclein aggregates in PD. In particular, impairements in the macroautophagy-lysosomal pathway (here referred to as autophagy), which is involved in the degradation of large cytosolic components, were found to promote α-synuclein aggregation. In contrast, autophagic stimulation has been shown to benefit α-synuclein degradation and rescue PD phenotypes in cell and rodent models. In this study, I examined the role of FYCO1 in modulating neuronal autophagic processes for α-synuclein aggregate clearance in hiPSC-derived neurons. FYCO1 is an interaction partner of the central autophagic regulator RAB7 but was mostly unnoticed since it was not found detrimental to cellular homeostasis under basal conditions. Still, previous work of our group has identified FYCO1 to rescue PD phenotypes in model systems such as HEK cells and Drosophila, due to improved α-synuclein clearance following FYCO1 overexpression. Mechanistically, FYCO1 is involved in autophagosome-lysosome fusion events by binding to autophagic vesicles, which is required for autophagosome maturation and final degradation. In addition, FYCO1 affiliates autophagic vesicles with the cellular transport machinery via kinesin motor proteins. While fusion promotion can be assigned to an enhancing effect on autophagic clearance, FYCO1-induced anterograde transport promotion is opposite to the retrograde trafficking route of autophagic vesicles for maturation, which is of special importance in neuronal axons. Here, I illuminated FYCO1 effects on both axonal vesicle transport processes and somal vesicle pools to evaluate its ability to promote autophagy-related degradation in neurons. To this end, I established a lentiviral transduction-based model in hiPSC-derived neurons to express FYCO1 in the presence of either a fluorescently labelled marker for autophagic vesicles (LC3-TFL) or in the presence of α-synuclein. In neuronal axons, FYCO1 overexpression impaired retrograde autophagic transport resulting in less movement, implying an inhibitory effect on axonal autophagy. In contrast, FYCO1 enhanced autophagic processes in neuronal somata by upregulating LC3 levels, promoting the collection of α-synuclein in autophagic vesicle clusters and increasing the colocalisation of autophagosomes with lysosomal markers, pointing to the advance in autophagosome maturation. I could not fully resolve, whether α-synuclein degradation was promoted by this induction, as α-synuclein clearance was not indicated yet in the time course of three weeks. Still, studying mutant forms of FYCO1 revealed deficits in autophagosome maturation, which were not represented with wild-type FYCO1. In particular, the autophagosome-interaction domain was essential for autophagosome-lysosome fusion and additionally seemed to be relevant for autophagosomes entering axonal transport, while mutations in the kinesin binding domain caused autophagosome acidification impairments. The most pronounced effect of FYCO1 overexpression in neurons was the modulation of lysosomal vesicles. Besides increasing lysosomal localisation to autophagic vesicles, FYCO1 promoted retrograde trafficking of axonal lysosomal vesicles, by a so far unresolved mechanism. As increasing transport of lysosomes toward the neuronal soma can be connected to the upregulation of autophagy, I hypothesise FYCO1 to be a mediator in autophagy induction signalling. Nevertheless, such an effect needs to be verified in future studies. Conclusively, with this work, I contributed to the understanding of FYCO1’s role in enhancing neuronal autophagic processes but further studies in more advanced PD models are required to evaluate whether this could contribute to an increased clearance of α-synuclein aggregates.

Parkinson’s disease (PD) is the second-most common neurodegenerative disease after Alzheimer’s disease. With 500,000 individuals currently living with Parkinson’s and nearly 60,000 new cases diagnosed each year, this disease causes significant financial burden on the healthcare system - amassing to annual expenditures totaling 200 billion dollars; predicted to increase through 2050. The disease phenotype is characterized by a combination of a resting tremor, bradykinesia, muscular rigidity, and depression due to dopaminergic neuronal death in the midbrain. The cause of the neurotoxicity has been largely discussed, with strong evidence suggesting that the protein, alpha-Synuclein, is a key factor. Under native conditions, alpha-Synuclein can be found localized at synaptic terminals where it is hypothesized to be involved in vesicle trafficking and recycling. However, its biochemical profile reveals a hydrophobic region that, once subjected to insult, initiates an aggregation cascade. Oligomeric species—products of the aggregation cascade—demonstrate marked neurotoxicity in dopaminergic neurons and illustrate migratory potential to neighboring healthy neurons, thereby contributing to progressive neurodegeneration. The current golden standard for PD diagnostics is a highly qualitative system involving a process-by-elimination with accuracy that is contingent upon physician experience. This, and a lack of standardized clinical testing procedures, lends to a 25% misdiagnosis rate. Even under circumstances of an accurate PD diagnosis, the only treatment options are pharmacologics that have a wide range of adverse side effects and ultimately contribute to systemic metabolic dysfunction. Thus, the research presented in this thesis seeks to overcome these current challenges by providing (1) a quantitative diagnostic platform and (2) a biomolecular therapeutic, towards oligomeric alpha-Synuclein. Aim 1: serves as a proof-of-concept for the use of catalytic nucleic acid moieties, deoxyribozymes and aptamers, to quantify alpha-Synuclein in a novel manner and explore the ability to detect oligomeric cytotoxic species. The cost-effective nature of these sensors allows for continued optimization. Aim 2: serves to establish a potential therapy that can abrogate alpha-synuclein oligomerization and toxicity through use of a modified Protein Disulfide Isomerase (PDI) peptide when introduced to live cells treated to simulate pre-parkinsonian pathology.