Protein misfolding and the accumulation of insoluble aggregates is a hallmark of several neurodegenerative disorders, including Alzheimer’s (AD) and Parkinson’s disease (PD). In AD and PD patients, extracellular protein deposits consisting of amyloid beta (Aβ) and intraneuronal inclusions composed of alpha synuclein (αS) are observed, respectively. Notably, the spatiotemporal patterning of soluble protein oligomers of αS and Aβ closely follow disease progression, giving support to an emerging role of soluble oligomers in PD and AD pathogenesis. However, the structural features underlying the toxicity of Aβ and αS oligomers remain elusive. This doctoral dissertation aims at elucidating the structural determinants of oligomer toxicity by focusing on the development and application of multidisciplinary approaches based primarily on solution NMR in combination with electron microscopy, multi-angle light scattering, fluorescence microscopy, wide-angle x-ray diffraction and cellular biophysics. Using this interdisciplinary approach, in chapters 2 and 3, we identify at atomic resolution the key structural elements that facilitate the colocalization, interaction and subsequent insertion of soluble Aβ oligomers into membranes, which ultimately result in the loss of membrane integrity. Notably, we show that small molecules, such as green tea catechins, remodel these structural features and effectively perturb the interactions with membranes. In chapter 4, we extend these analyses to αS and identify how the chaperone, Human Serum Albumin (HSA), remodels toxic αS oligomers into non-toxic species and breaks the catalytic cycle that generates new toxic oligomers. Lastly, in chapter 5, we describe a novel solution NMR approach to map at atomic resolution the sites of early self-association, with minimal bias from monomer dynamics, an effect that frequently dominates residue-dependent variations in solution NMR measurements. Overall, given that Aβ and αS are archetypical amyloidogenic proteins, we anticipate that the structure – toxicity relationships established herein, and the related experimental approaches may be transferrable to other amyloidogenic systems. / Dissertation / Doctor of Philosophy (PhD)

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Pesticides have been suggested to play a role in the development of many neurodegerative diseases including Parkinson's disease and Alzheimer's disease. Additionally, it has been suggested that exposure to pesticides and other environmental chemicals during the early stages of life could result in an increased vulnerability to such substances that could lead to neurotoxicity and degeneration late in life. We hypothesized that exposure to mixtures of certain pesticides could change neurotransmitter levels and cellular oxidative stress and that this would be greater in mice exposed early and later in life than mice exposed only as adults. We studied the effects of permethrin (PR) (a pyrethroid type I) and endosulfan (EN) (an organochlorine) on the levels of catecholamines, indolamines, acetylcholinesterase, lipid peroxidation and α-synuclein in the brain of mice. These pesticides have different structures but both are known to modify the kinetics of voltage-sensitive ion channels and calcium ion flux/homeostasis that could affect the release of several neurotransmitters. The study consisted of two experiments: In the first experiment, adult C57Bl/6 mice (7-9 months old) were injected, intraperitoneally, with the following treatments: EN 4.3, 2.15 mg/kg; PR 150, 15 mg/kg and their mixtures EN 4.3 + PR 150 and EN 2.15 + PR 15 mg/kg. Mice were sacrificed 24 hrs after the last injection. In the second experiment, doses consisted of EN 0.7, 1.4 mg/kg, PR 1.5, 15 mg/kg and their mixtures EN 0.7 + PR 1.5 mg/kg and EN 1.4 + PR 15 mg/kg were given to juvenile mice intraperitoneally daily during a period of two weeks from postnatal day 5 to 19. Mice were then, left undisturbed with their dams. Re-challenge was performed when mice were 7-9 months old and dosages of EN 4.3, 2.15 mg/kg, PR 150, 15 mg/kg and their mixtures, EN 4.3 + PR 150 and EN 2.15 + PR 15 mg/kg were given intraperitoneally every other day during a period of two weeks to match the treatments when pesticide exposure was only as adults. Mice were sacrificed 24 hrs after the last injection. The corpora striatum was extracted and analyzed by HPLC for catecholamines (dopamine, DOPAC, homovalinic acid and norepinephrine) and indolamines (serotonin and 5-HIAA). In general low doses of permethrin and endosulfan alone and in combination (EN 2.15 + PR 15 mg/kg) altered the levels of catecholamines and indolamines in both studies with adult mice and mice dosed as juveniles and re-challenged as adults. Catecholamine and indolamines levels were affected to a greater extent in the adult mice than in mice dosed as juveniles and re-challenged as adults, when compared to controls. Acetylcholinesterase was increased under both exposure situations but again adult mice seemed to be more affected than mice dosed as juveniles and re-challenged as adults. Because reactive oxygen species have been implicated in the development of Parkinson's disease, and are known to cause degradation of certain neurotransmitters, we monitored the levels of lipid peroxides in brain cortex as an indicator of free radical tissue damage. The peroxide levels were measured by thiobarbituric acid reactive products (TBARS). Increased levels of lipid peroxides were significant in the low dose treatment groups of the adult study. However, there seemed to be a pattern between the levels of dopamine and DOPAC in the striatum and the levels of peroxidation in cortex. The presence of dopamine metabolites appeared to be related to high levels of peroxidation within the basal ganglia and up-regulation of proteins such as α-synuclein. Western blots of α-synuclein in both experiments of the study showed intense double and triple bands that corresponded to aggregated α-synuclein. In general, when compared with controls, mice dosed as juveniles and re-challenged as adults did not alter the above parameters as much as mice dosed only as adults. Instead, the mice first dosed as juveniles seemed to develop an adaptation response to the later exposure of these pesticides. Taking all these results into account, early exposure and re-challenge with permethrin and endosulfan in this study appeared to induce a protective response against neurochemical changes in the brain of these mice. In addition, low doses of these pesticides and the low dose combination mixture seem to exert an effect on the parameters studied. Therefore, exposure to pesticides such as endosulfan and permethrin and their combinations could make a contribution towards the initiation or aggravation of biochemical neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. / Master of Science

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease. The main symptoms of PD include tremor, muscle stiffness, slowness of movement, and postural instability. Motor symptoms of PD are driven by pathology primarily in the substantia nigra (SN) region of the brain, where there is dopaminergic neuron death and a-Synuclein (a-Syn) protein aggregation. Simultaneously, there is persistent inflammation in the central nervous system (CNS) and in the periphery of PD patients that begins prior to the onset of motor symptoms. Animal models of PD can exhibit inflammation in the brain and periphery, but the role of the immune system in disease pathogenesis and progression is not fully understood. In Chapter 1, I introduce PD neuropathology, genetic and environmental risk factors, and hypotheses for a-Syn aggregation and propagation from the periphery to the brain. Then, I discuss the altered immune responses and features of autoimmunity in PD patients. Lastly, I describe animal models of PD and how they are used to investigate the role of the immune system in PD pathology. Features of autoimmunity against the a-Syn protein are present in PD patients and appear prior to the onset of motor symptoms. Whether autoimmunity to a-Syn can alone initiate PD pathology has not been tested in animal models of PD. In Chapter 3, I demonstrate that activation of a-Syn32-46-specific CD4+ T cells in transgenic humanized mice induced early features of PD in the gut. We found that humanized mice immunized with a-Syn32-46 experience constipation, a common early PD symptom, as well as enteric neuron loss. Further investigation into the immune response in the gut revealed activated innate and adaptive immune responses which promoted dopaminergic neuron loss in the small intestines. PD pathology, however, did not develop in the brain, suggesting that autoimmunity to a-Syn32-46 promotes pathology at prodromal PD stages. Elevated T cell infiltration is found in the PD patient brain, suggesting that the adaptive immune response may also promote pathology in the brain at later disease stages. I hypothesized that a-Syn-specific CD4+ T cells may promote PD neuropathology upon a-Syn aggregation. In Chapter 4, I tested this hypothesis in both humanized and wild-type mice. I found that active immunization with a-Syn32-46 in humanized mice combined with human a-Syn overexpression in the brain did not increase T cell CNS infiltration. However, adoptive transfer of a-Syn-specific T cells into wildtype mice that overexpress human a-Syn increases T cell CNS infiltration and CNS myeloid cell activation. These results suggest that a-Syn-specific T cells may promote neuroinflammation in the presence of a-Syn accumulation. In Chapter 5, I summarize the major findings from my research and future directions which can provide a more detailed understanding of the ways that a-Syn-specific T cells promote PD neuropathology. Furthermore, I discuss innovations in the diagnosis and treatment of PD and the potential for immunotherapies to significantly modify disease course.

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In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.

In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.

<p dir="ltr">My research projects focuses on the dual targeting of small molecules to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to reduce the spread of AD and related dementias. Not very many drug discovery programs focus on the specific isoforms of the tau protein. We established two series of compounds: aminoindole compounds connected by a carboxamide and triazine compounds connected by a triazine linker. Using biophysical methods we evaluated the effectiveness of both series of compounds in decreasing the amount of misfolded α-syn and tau protein in order to explore their anti-aggregation potential.</p><p><br></p>

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are defined by neurodegeneration and accumulations of misfolded proteins that spread through the brain in a well characterized manner. In AD these accumulations consist mainly of β-amyloid (Aβ) and tau, while in PD, α-synuclein (α-syn) make up the characteristic lewy pathology.     The general aim of this thesis was to investigate mechanisms associated with neurotoxic peptide activity by Aβ, tau and α-syn in relation to cellular degradation and transfer with a cell-to-cell transfer model system.    We found that intercellular transfer of oligomeric Aβ occurs independently of isoform. However, the amount of transfer correlates with each isoforms ability to resist degradation or cellular clearance. The Aβ1-42 isoform showed particular resistance to clearance, which resulted in higher levels of cell-to-cell transfer of the isoform and lysosomal stress caused by accumulation.    As Aβ accumulations can inhibit the proteasomal degradation we investigated how reduced proteasomal degradation affected neuron-like cells. We found increased levels of phosphorylated tau protein, disturbed microtubule stability and impaired neuritic transport after reduced proteasomal activity. These changes was partly linked to c-Jun and ERK 1/2 kinase activity.    We could also show that α-syn transferred from cell-to-cell in our model system, with a higher degree of transfer for the larger oligomer and fibrillar species. Similar to Aβ, α-syn mainly colocalized with lysosomes, before and after transfer.     Lastly, we have developed our cell-to-cell transfer system into a model suitable for high throughput screening (HTS). The type of cells have been upgraded from SH-SY5Y cells to induced pluripotent stem cells (iPSCs), with a differentiation profile more similar to mature neurons. The next step will be screening a small molecular library for substances with inhibitory effect on cell-to-cell transfer of Aβ peptides.     The importance of the degradative systems in maintaining protein homeostasis and prevent toxic accumulations in general is well known. Our findings shows the importance of these systems for neurodegenerative diseases and also highlight the link between degradation and cell-to-cell transfer. To restore or enhance the degradative systems would be an interesting avenue to treat neurodegenerative diseases. Another way would be to inhibit the transfer of misfolded protein aggregates. By using the HTS model we developed, a candidate substance with good inhibitory effect on transfer can hopefully be found.