The brain is a highly complex structure; neurons extend axons which follow precise paths to make connections with their targets. This extension is guided by a specialised and highly motile structure at the axon tip -the growth cone- which integrates guidance cues to steer the axon through the environment. Aberrant pathfinding is likely to result in developmental impairments causing disruption to brain functions underlying emotion learning and memory. Furthermore, pre-existing connections are constantly remodelled, the ability to do so declines with age, and can have huge impacts on quality of life and well-being. Examining how changes in growth cone behaviour triggered by external cues occurs is crucial for understanding processes in both development and disease. Controlled reorganisation of growth cone cytoskeletal components, such as actin filaments, generate membrane protrusions forming lamellipodia and filopodia. Filopodium formation is commonly associated with sensing the mechanical and chemical environment of the cell. Despite our understanding of the guidance choices that can be made, how filopodia transmit information at a molecular level leading to profound changes in morphology, motility and directionality remains largely unknown. Various actin-binding proteins regulate the number, stability and branching of filopodia. They may therefore have a key role in priming or abrogating the ability of the growth cone to respond to a given guidance cue. I have shown that the actin binding proteins drebrin and cofilin, whilst displaying opposing molecular activities on actin filaments, work synergistically in a temporally regulated manner. A fluorescent membrane marker combined with tagged cofilin and drebrin enabled accurate correlation of cofilin and drebrin dynamics with growth cone morphology and filopodial turnover in live neurons. In contrast to previous in vitro experiments, cofilin was found to enhance the effect of drebrin to promote filopodia formation in intact neurons, and that growth cone spread was significantly constrained when cofilin was knocked down. Importantly, this adds to our understanding of how the two actin binding proteins contribute to directed motility in neuronal growth cone filopodia during guidance. Furthermore, following acute treatment with low concentrations of the repulsive guidance cue semaphorin-3A, neuronal growth cones expressing cofilin displayed increased morphological complexity and filopodial stability. This suggests that traditional collapse signals may serve as pause signals allowing neurons to increase the surface area to sense the environment adequately and enable precise wiring decisions. Remodeling of the cytoskeleton is perturbed in a number of degenerative diseases including Alzheimer's, Huntington's, and Amyotrophic Lateral Sclerosis. These conditions are associated with widespread synaptic loss, resulting in memory loss, cognitive impairment, and movement disorders which leads to severe deterioration in quality of life for those afflicted in addition to wider negative socioeconomic impacts. How widespread synaptic loss occurs is poorly understood. One common characteristic is neuronal stress which can be initiated through different conditions such as neuroinflammation, energetic stress, glutamate excitotoxicity, and accumulation of misfolded proteins, all of which have been associated with perturbation of the actin cytoskeleton and the initiation of the cofilin-actin rod stress response. Dysfunction of the cytoskeleton can lead to the disruption of synaptic activity by blocking the delivery of elements such as organelles and proteins required for maintenance of the synapse. Modulating this stress response offers an approach to protecting the integrity of normal synaptic function. Actin interacting protein-1 is a conserved actin binding protein that enhances the filament disassembly activity of cofilin. I have discovered that AIP-1 has a potent ability to prevent the formation of cofilin rods which are thought to contribute to the neuronal dysfunction in several neurodegenerative disorders, even when they are treated with amyloid-β or subjected to metabolic stress. This is the first study to demonstrate a molecular mechanism for preventing rod formation in the presence of a neuronal stressor and has the potential to protect against rod formation by other stressors associated with disease such as inflammation and excitotoxicity. AIP-1 offers the exciting possibility of a means to reverse cofilin rod formation and the subsequent cytoskeletal pathology associated with dementia and has potential for therapeutic exploitation in human disease. Furthermore, it is the first study to demonstrate that AIP-1 localises to areas of rapid actin remodeling in neuronal growth cones. Exploiting the action of AIP-1 therefore represents an exciting and novel therapeutic avenue to tackle neurodegeneration.

A inflamação é fundamental para a manutenção da homeostasia e para a resposta do organismo à injúria. A resposta inflamatória deve ser adequada aos estímulos agressores; no sistema nervoso central, sua inadequação conduz à gênese de diferentes doenças neurodegenerativas. A proteína anexina A1 (ANXA1) e os receptores ativados por proliferadores de peroxissomo (PPAR) controlam a inflamação, pois ambos inibem o desenvolvimento da inflamação e aceleram sua resolução. Nosso grupo de pesquisa tem mostrado que a ANXA1 modula a expressão de PPARγ em macrófagos. Assim, o presente trabalho investigou a modulação da expressão do PPARγ e das suas funções em células da microglia pela ANXA1. Foram empregadas células imortalizadas da linhagem BV2 (microglia murina), inalteradas ou transfectadas para redução da expressão de ANXA1, tratadas com ANXA1 exógena (recombinante - rANXA1) ou com agonista ou antagonista de PPARγ (pioglitazona e GW9662, respectivamente). Os resultados obtidos mostraram que: 1) tratamento com rANXA1 aumenta as expressões gênica (RT-PCR) e proteica (Western Blotting) de PPARγ, e ambas as expressões estão reduzidas em células com deficiência endógena de ANXA1, sendo que tal efeito foi revertido pela ação da rANXA1; 2) tratamento com rANXA1 não induz a expressão dos fatores de transcrição ligados a expressão de PPARγ: proteínas ligantes de elementos de resposta ao cAMP - CREB - e transdutores de sinais e ativadores de transcrição - STAT6 - (Western Blotting), mas os níveis de ambos os fatores estão reduzidos em células transfectadas, e tal efeito não foi revertido pelo tratamento com rANXA1; 3) tratamento com pioglitazona ou com rANXA1 individualmente aumenta a fagocitose de células PC12 apoptóticas (citometria de fluxo), mas o tratamento simultâneo não altera a fagocitose induzida por pioglitazona ou rANXA1; no entanto, tratamento com GW9662 inibiu a fagocitose induzida pelo tratamento com rANXA1; 4) o tratamento com rANXA1 aumenta a expressão de CD36 (citometria de fluxo); a expressão de CD36 está reduzida em células transfectadas e tal expressão não é revertida pelo tratamento com rANXA1. Em conjunto, os dados obtidos mostram a modulação da ANXA1 sobre PPARγ em células da micróglia, com possível ação sobre a fagocitose de células apoptóticas, e que a redução da expressão de ANXA1 reduz acentuadamente a expressão dos fatores de transcrição STAT6 e CREB, bem como a expressão de CD36. A elucidação dos efeitos resultantes destas alterações desencadeadas pela deficiência de ANXA1 endógena poderá contribuir para compreensão da fisiopatologia da neuroinflamação. / Inflammation is a key process in maintaining homeostasis and is essential for the body\'s response to injury. The inflammatory response must be proportional to the aggressor stimuli; in the central nervous system, a failed proper modulation leads to the development of different neurodegenerative diseases. Protein annexin A1 (ANXA1) and peroxisome proliferated-activated receptors (PPAR) control inflammation, as both inhibit development of inflammation and accelerate its resolution. Our research group has demonstrated that ANXA1 modulates PPARγ expression in macrophages. Thus, the present work investigated the modulation of PPARγ expression and its functions in microglia cells by ANXA1. In order to assess such, immortalized cells from cell line BV2 (murine microglia), either unadulterated or transfected for reduced expression of ANXA1, were treated with exogenous ANXA1 (recombinant protein - rANXA1) or either with PPARγ agonist or antagonist (pioglitazone and GW9662, respectively). The obtained results demonstrated that: 1) treatment with rANXA1 increases both gene (RT-PCR) and protein (Western Blotting) expressions of PPARγ, and also that both expressions are reduced in cells with endogenous deficiency of ANXA1, and such effect was reversed by the actions of rANXA1; 2) treatment with rANXA1 does not promote the expression of transcription factors associated with PPARγ expression: cAMP response element binding protein - CREB - and signal transductor and activator of transcription 6 - STAT6 (Western Blotting), but the expression levels of both factors are reduced in transfected cells, and such effect was not reversed by treatment with rANXA1; 3) individual treatment with pioglitazone or rANXA1 increases phagocytosis of apoptotic PC12 cells (flow cytometry), but simultaneous treatment does not affect pioglitazone/rANXA1-induced phagocytosis; however, treatment with GW9662 inhibited rANXA1-induced phagocytosis; 4) treatment with rANXA1 increases CD36 expression (flow cytometry); the expression of CD36 is reduced in transfected cells, and such expression is not reversed by treatment with rANXA1. The obtained data demonstrate the modulation ANXA1 exerts upon PPARγ in microglia cells, with a possible action upon phagocytosis of apoptotic cells, and that reduction of ANXA1 expression greatly reduces the expression of transcription factors STAT6 and CREB, as well as the expression of CD36. Elucidation of such effects that arise from a deficiency of endogenous ANXA1 will contribute to a better comprehension of the pathophysiology of neuroinflammation.

O uso da cocaína na forma de crack vem crescendo nos últimos anos quando comparado às demais vias de administração. Contribuem para esse fato a obtenção quase imediata de efeitos e a maior facilidade de uso, que dispensa a necessidade de material injetável. O usuário de crack sofre os efeitos não só da cocaína, mas também de seu produto de pirólise, a metilecgonidina (AEME). Existem evidências de que a cocaína leva à neurodegeneração, entretanto a participação da AEME nesse processo ainda não foi estudada. A proposta deste estudo foi investigar a participação da AEME no processo neurodegenerativo utilizando cultura primária de hipocampo realizada a partir de fetos de ratos. Foram realizados ensaios de viabilidade celular (MTT) e da atividade da lactato desidrogenase (LDH), além da avaliação morfológica por microscopia de fluorescência. Foi estudada também a participação do dano oxidativo no processo de neurodegeneração, como a formação de aduto de DNA; atividade das enzimas antioxidantes glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa S-transferase (GST); e a produção de malonaldeído (MDA), um biomarcador de peroxidação lipídica. Tanto a cocaína quanto a AEME mostraram-se neurotóxicas. A partir dos ensaios de viabilidade e da avaliação morfológica foi possível inferir que, em células hipocampais, a cocaína leva à morte celular tanto por necrose quanto por apoptose e que a provável via envolvida na neurodegeneração da AEME é a apoptose. A AEME não causou lesão direta ao DNA, uma vez que não foi observada a formação de adutos nem com a desoxiguanosina (d-G), a base nitrogenada mais reativa, nem com DNA comercial. Mais ainda, nossos resultados mostraram que a AEME e a cocaína, nas concentrações de 1 e 2 mM, respectivamente, foram equipotentes e a incubação concomitante das duas substâncias nessas concentrações apresentou efeito aditivo após 48 horas de exposição. A morte de células hopocampais evidenciada a partir de 24 horas de exposição foi precedida pela diminuição da atividade da GPx após 3 horas de incubação tanto com a AEME e a cocaína, quanto com a associação entre essas substâncias. A atividade da GST também diminuiu, no entanto, somente após 6 horas de exposição, antecedendo a morte celular. Não foi observada alteração na atividade da GR. Houve um aumento, porém, não estatisticamente significativo, de MDA após 48 horas de incubação. Nossos resultados sugerem uma maior susceptibilidade à neurodegeneração com o uso de crack do que com a cocaína isoladamente. / Smoking crack has increased in the last years when compared to the other routes of cocaine administration. Its advantage is the quicker and stronger high effects and its ease of use without the need of needles. Smoking crack involves inhaling not only cocaine, but also its pyrolysis product, methylecgonidine (AEME). There are evidences that cocaine causes neurodegeneration, but AEME involvement in this process has not been studied yet. The aim of this study was to investigate AEME participation in neurodegeneration using a primary hippocampus culture made from rat fetuses. Cellular viability assays (MTT), lactate dehydrogenase activity (LDH) and morphological evaluations with fluorescence microscopy were performed. The involvement of oxidative injury in the neurodegeneration process was also studied through DNA adduct formation; the evaluation of antioxidants enzymes activities as glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST); and the production of malonaldehyde (MDA), a lipoperoxidation biomarker. Both cocaine and AEME showed neurotoxic effects. Through viability assays and morphologic evaluations we can suggest that, in hippocampal cells, cocaine cell death mechanism involves not only necrosis, but also apoptosis and that AEME pathway involved in neurodegeneration is only apoptosis. AEME did not produce a direct DNA injury, as no DNA adduct was observed with desoxyguanosine (d-G), the most reactive nitrogenous base, nor with commercial DNA. Moreover, our results showed that 1 and 2 mM of AEME and cocaine, respectively, were equipotent and the concomitant incubation of both compounds in those concentrations showed additive effect after 48 hours of exposure. Hippocampal cell death at 24 hours was preceded by a decrease in GPx activity after 3 hours of incubation with AEME, cocaine and association between these two compounds. GST activity also decreased but only after 6 hours of exposure, also before cell death. There was no alteration in GR activity. There was an increase, although not statistically significant, in MDA after 48 hours of exposure. As smoking crack abusers are exposed to both cocaine and AEME, our results suggest a higher susceptibility to neurodegeneration in smoking crack than with cocaine alone.

Rho GTPases and their regulators such as guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) represent an important class of molecules controlling dendritic spine plasticity. Although they are typically described as cytoskeletal modulators, roles for the GTPases in endocytosis and cell polarity establishment have also been defined. The neuronal proteins a1- and a2-chimaerins belong to a group of Rac and Cdc42 GAPs that inactivate these GTPases; in addition to a GAP domain, the a-chimaerins share a phosphokinase C (PKC)-like C1 domain but have distinct N-terminal domains (NTDs). My project has explored the importance of specific domains of a1-chimaerin both in induction of a morphological cellular protrusion collapse phenotype ('circularisation') and in interactions with partner proteins that may help to explain the phenotype. The results described in my thesis show that a1-chimaerin possesses a previously undescribed C-terminal domain (CTD) that is indispensable for the ability of the protein to induce collapse of protrusions, and consequent circularisation, in various cell types; moreover, an intact CTD is also important for association of a1-chimaerin with its known effector EphA4, and potentially with other undefined membrane proteins, in a C1-domain- dependent manner. In addition, my results show that a1-chimaerin associates via its NTD with the Src kinase Fyn, and via its C1 domain with the NR2A subunit of the NMDA receptor. Further experiments explored a1-chimaerin effects on EphA4 and NMDA receptor cell surface expression, as well as binding to other putative partners - including the adaptor protein p35 and the polarity protein PAR6. Finally, I have shown that inhibition of a pathway involving the Rho-associated coiled-coil containing protein kinase (ROCK) reverts circularisation induced by a1- chimaerin, and that a blocking peptide based on the CTD may be employed to partially counteract the phenotype. These results uncover a novel domain in a1-chimaerin that may have a crucial importance for the induction of cellular process collapse by a1-chimaerin with a potential relevance to the EphA4-induced dendritic spine retraction, EphA4 receptor endocytosis, and cell surface expression of NR2A-containing NMDA receptors. This suggests a model of a multi-protein signalling complex involving a1-chimaerin that coordinates cellular process remodelling, and that is likely to be important both for adult neuronal circuit plasticity and for neurodegenerative diseases.

Ce travail de thèse porte sur l'étude de la réaction microgliale et des interactions microglie/neurones dans un modèle murin de neurodégénérescence dopaminergique induit par l'injection de 6‐hydroxydopamine (6‐ OHDA). Dans ce modèle, nous décrivons tout d'abord les cinétiques d'activation microgliale, de perte neuronale et d'altérations comportementales en relation avec le déficit dopaminergique. Dans la substance noire lésée ont été observées une perte progressive des neurones dopaminergiques TH+ (Tyrosine Hydroxylase) ainsi qu'une activation microgliale précoce mais transitoire. Le rôle délétère de cette activation microgliale est fortement suggéré par la mise en évidence d'une protection partielle contre la toxicité induite par la 6‐OHDA dans des souris génétiquement modifiées DAP12 Knock‐In, dont la densité microgliale est constitutivement diminuée. Par ailleurs, nous avons identifié différents types de contacts intercellulaires entre les neurones et la microglie de la substance noire lésée. Ces interactions physiques sont matérialisées entre autres sous la forme de contacts intimes entre le corps cellulaire des cellules microgliales et le soma des neurones dopaminergiques. De façon intéressante, ce type d'interaction se met en place quelques jours avant le pic de mort neuronale et dans la grande majorité des cas, concerne des neurones présentant des signes morphologiques d'apoptose. Finalement, nous avons également identifié un nouveau type d'interaction physique entre neurones et microglie sous la forme de ramifications microgliales pénétrant le soma des neurones. Ces interactions s'apparentent aux "tunelling nanotubes" décrits dans la littérature et représentent un type particulier de ramifications microgliales perforantes que nous avons nommées "tunelling ramifications". La présence de vacuoles TH+ dans le cytoplasme de nombreuses cellules microgliales suggère que les ramifications microgliales pénétrantes sont le support d'un processus de microphagocytose ciblant le cytoplasme des neurones dopaminergiques. La fonction précise de ces interactions et les mécanismes moléculaires qui les suscitent restent à définir. Toutefois, ce travail de thèse apporte un ensemble de données originales sur le dialogue microglie/neurones dans un modèle murin de la maladie de Parkinson / This thesis work is aimed to study microglial reaction and microglia/neuron interactions in a murine model of dopaminergic neurodegeneration induced by the injection of 6‐hydroxydopamine (6‐OHDA). In this model, we first describe the kinetics of microglial activation, neuronal cell loss and behavioral alterations in relation with the dopaminergic defect. In the injured substantia nigra, we observed a progressive loss of TH+ (Tyrosine Hydroxylase ‐positive) dopaminergic neurons and an early but transient microglial activation. The deleterious role of microglial activation is strongly suggested by the observation of a partial neuroprotection against 6‐OHDA‐induced toxicity in genetically DAP12 Knock‐In mice, in which microglial cells are defective in regard to their number and function. In addition, we identified various types of cell‐tocell contacts between neurons and microglia in the injured substantia nigra. Such physical interactions were established between microglia and neuronal cell bodies several days before the peak of neuronal death and in the majority of cases in neurons showing morphological signs of apoptosis. Finally, we also identified a new type of physical interactions consisting in microglial ramifications penetrating the soma of TH+ neurons. These interactions present similarities with the so‐called « tunelling nanotubes » previously described in the literature and represent a particular type of penetrating microglial ramifications the we named "tunelling ramifications.". Interestingly, in the injured substantia nigra, the presence of TH+ vacuoles in the cytoplasm of numerous microglial cells strongly suggests that microglial ramifications support microphagocytosis targeted toward the cytoplasm of dopaminergic neurons. The precise function and molecular mechanisms of such unique interactions need to be further assessed. However, our work provides a set of original data that deepens our knowledge on the dialogue between microglia and neurons in a mouse model of Parkinson's disease

Ce travail de thèse décrit et analyse la réaction neuro-inflammatoire accompagnant la mort cellulaire neuronale dans un modèle murin de la maladie de Parkinson. Dans ce modèle, induit par l’injection intrastriatale d'un analogue toxique de la dopamine, la 6-hydroxydopamine (6-OHDA), nous décrivons les caractéristiques et la cinétique de l’activation microgliale, de l'infiltration lymphocytaire T, de la perte de neurones dopaminergiques TH+ (Tyrosine Hydroxylase) et des altérations du comportement moteur. Nos observations sont complétées par une étude neuropathologique de la substance noire chez des patients atteints de maladie de Parkinson. Les résultats montrent que, chez l'homme comme chez la souris, la mort de neurones dopaminergiques induit une infiltration T de faible intensité, limitée à la substance noire et s'accompagnant d'une activation microgliale. Dans un deuxième temps, nous analysons l'impact d'une déficience lymphocytaire T génétiquement déterminée sur les paramètres histologiques et fonctionnels caractérisant le modèle 6-OHDA. Nos résultats montrent que, comparées à des souris contrôles immunocompétentes, les souris immunodéficientes de souche Foxn1 KO, CD3 KO, NOD SCID ou RAG1 KO présentent toutes, à des degrés divers, une susceptibilité significativement accrue aux effets neurotoxiques de la 6-OHDA. L'aggravation observée de la perte neuronale s'accompagne d'une accentuation majeure des troubles du comportement moteur et de l'activation microgliale. Ce travail démontre l'importance de la neuro-inflammation et de l'immunité adaptative dans la physiopathologie du modèle 6-OHDA. Il suggère également que les LyT infiltrant la substance noire des patients atteints de maladie de Parkinson exercent un rôle inhibiteur sur l'activation microgliale et pourraient par ce mécanisme ralentir l'évolution de la perte neuronale dopaminergique. En résumé, ce travail de thèse apporte un ensemble de données originales sur les interactions entre LyT, microglie et neurones dopaminergiques dans le contexte de la maladie de Parkinson et du modèle murin 6-OHDA / This thesis work describes and analyzes the neuroinflammatory reaction that accompanies neuronal cell death in a murine model of Parkinson's disease. In this model, induced by the intrastriatal injection of 6-hydroxydopamine (6-OHDA), a toxic dopamine analog, we report on the main features and kinetics of microglial activation, T-cell infiltration, loss of TH+ (Tyrosine Hydroxylase) dopaminergic neurons and motor behavior alterations. We also assessed the presence of T-cells in the susbstantia nigra of Parkinson's disease patients and found that, as observed in the 6-OHDA murine model, the neuronal cell death of dopaminergic neurons triggers a low-grade T-cell infiltration that accompanies microglial activation. We then studied the impact of genetically-determined T-cell immunodeficiency on histological and functional outcomes in the 6-OHDA model. Our results show that, as compared to immunocompetent control mice, immunodeficient strains consisting in Foxn1 KO, CD3 KO, NOD SCID or RAG KO mice consistently presented, at varied levels, a highest susceptibility to 6-OHDA induced dopaminergic neurodegeneration. The observed accentuation of neuronal cell loss was accompanied by a marked increase of microglial activation and motor behavior alterations. Our work demonstrates the pathophysiological role of neuroinflammation and adaptative immunity in the 6-OHDA model. It also suggests that T-cells infiltrating the substantia nigra of Parkinson's disease patients dampen microglial activation and could, via this inhibitory effect, slow the progression of dopaminergic cell loss. Overall this thesis work provides original data on the interactions between T-cells, microglia and dopaminergic neurons in the context of Parkinson's disease and the murine 6-OHDA model

This thesis work describes the role of Rab proteins in amyloid processing and clearance in different cell pathways. It also describes an experimental approach used to analyze the expression effects of Rab10 in amyloid beta production. Since the main theory behind neurodegeneration in Alzheimer's disease claims that high levels of amyloid beta 42 (Aβ42) molecules trigger widespread neuronal death, control of Aβ42 has been a main target in Alzheimer's disease research. In addition, several studies show increased levels of particular Rab proteins in Alzheimer's pathogenesis. However, no review consolidates current findings in neurodegeneration of Alzheimer's with Rab protein dysfunction. The first chapter of this thesis aims to address this need by providing a current review of Rab proteins associated with APP and neurodegeneration. The second chapter constitutes an experimental approach used to characterize the effects of Rab10 and Sar1A GTPases in APP and amyloid processing. We found that Rab10 expression does not affect APP production but significantly changes Aβ generation, particularly the toxic Aβ42 and Aβ42:40 ratio. On the other hand, we found no significant effect of Sar1A expression on either APP or amyloid beta generation. These findings partially confirm the work done by Kauwe et al (2015) and provide preliminary evidence for two potential targets for protective effects in neurodegeneration.

An estimated 13.9% of Americans currently meet criteria for an alcohol (ethanol; EtOH) use disorder (AUD). While there are 4 medications approved by the Food and Drug Administration (FDA) to treat AUD, these treatments have demonstrated poor clinical efficacy. Our ongoing research program encompasses a multi-tiered screening of a natural product library and validation process to provide novel information about the mechanisms underlying EtOH-induced changes in neurobiology and to identify novel chemical scaffolds to be exploited in the development of pharmacological treatments for AUD in a rodent organotypic hippocampal slice culture model. Initial screens of several natural product compounds identified 3 compounds which attenuate 48 h EtOH-induced cytotoxicity in vitro. As analogs of natural products can be developed to have enhanced therapeutic potential over parental structures, Study 1 sought to extend on prior findings via the screening of several natural product analogs for their ability to attenuate EtOH-induced cytoxicity. Nine natural produce analogs demonstrated potent cytoprotective effects against EtOH-induced toxicity at 48 h. Several reports suggest EtOH-induced neurotoxicity may be secondary to the induction of persistent neuroimmune activation, and isoflavonoids have been shown to have effects on neuroimmune signaling. Thus, Study 2 compared the effects of compound 9b, an isoflavonoid analog identified in Study 1, to daidzein (DZ), a prototypical isoflavonoid, in the same 48 h model, with the addition of a neuroimmune component. Specifically, culture media was collected to assess for the release of the neuroimmune mediators HMGB1, TNF-α, IL-6, and IL-10 via ELISA. Compound 9b and DZ protected against EtOH-induced cytotoxicity at 48 h. EtOH exposure significantly increased secretion of HMGB1 and IL-6 into culture media at 48h. Compound 9b and DZ attenuated these increases at all concentrations tested. These results suggest potential neuroimmune modulating properties of isoflavonoids which may contribute to their neuroprotective effects against EtOH in vitro. These findings highlight the potential applications DZ and the novel isoflavonoid analog 9b for use in the treatment of AUD.

Parkinson’s disease (PD) is a disorder of the nervous system that causes problems with movement (motor symptoms) as well as other problems such as mood disorders, cognitive changes, sleep disorders, constipation, pain, and other non-motor symptoms. The severity of PD symptoms worsens over time as the disease progresses, and while there are treatments for the motor and some non-motor symptoms there is no known cure for PD. Thus there is a high demand for therapies to slow the progressive neurodegeneration observed in PD. Two clinical trials at the University of Kentucky College of Medicine (NCT02369003, NCT01833364) are currently underway that aim to develop a disease-modifying therapy that slows the progression of PD. These clinical trials are evaluating the safety and feasibility of an autologous peripheral nerve graft to the substantia nigra in combination with Deep Brain Stimulation (DBS) for the treatment of PD. By grafting peripheral nerve tissue to the Substantia Nigra, the researchers aim to introduce peripheral nerve tissue, which is capable of functional regeneration after injury, to the degenerating Substantia Nigra of patients with PD. The central hypothesis of these clinical trials is that the grafted tissue will slow degeneration of the target brain region through neural repair actions of Schwann cells as well as other pro-regenerative features of the peripheral nerve tissue. This dissertation details analysis of the peripheral nerve tissue used in the above clinical trials with respect to tissue composition and gene expression, both of injury-naive human peripheral nerve as well as the post-conditioning injury nerve tissue used in the grafting procedure. RNA-seq analysis of sural nerve tissue pre and post-conditioning show significant changes in gene expression corresponding with transdifferentiation of Schwann cells from a myelinating to a repair phenotype, release of growth factors, activation of macrophages and other immune cells, and an increase in anti-apoptotic and neuroprotective gene transcripts. These results reveal in vivo gene expression changes involved in the human peripheral nerve injury repair process, which has relevance beyond this clinical trial to the fields of Schwann cell biology and peripheral nerve repair. To assess the neurobiology of the graft post-implantation we developed an animal model of the grafting procedure, termed Neuro-Avatars, which feature human graft tissue implanted into athymic nude rats. Survival and infiltration of human graft cells into the host brain were shown using immunohistochemistry of Human Nuclear Antigen. Surgical methods and outcomes from the ongoing development of this animal model are reported. To connect the results of these laboratory studies to the clinical trial we compared the severity of motor symptoms before surgery to one year post-surgery in patients who received the analyzed graft tissue. Motor symptom severity was assessed using the Unified Parkinson’s Disease Rating Scale Part III. Finally, the implications and future directions of this research is discussed. In summary, this dissertation advances the translational science cycle by using clinical trial findings and samples to answer basic science questions that will in turn guide future clinical trial design.

Amyloidoses comprise a group of gain-of-toxic function protein misfolding diseases, in which normally soluble proteins in their functional state undergo conformational changes into highly organized and generally intractable thread-like aggregates, termed amyloid fibrils. These structures accumulate predominantly in the extracellular space but growing evidence suggests that amyloids may start to form intracellularly. At least 26 different human proteins, intact or in fragmented form, are known to form amyloid, which is linked with many debilitating neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and transthyretin (TTR)-related amyloidosis (ATTR). In this work, we focus on ATTR, which is one of the most frequent systemic amyloid diseases. A functional link was established between hereditary ATTR, a severe and fatal disorder and the enhanced propensity of the human plasma protein transthyretin (TTR) to form aggregates, caused by single point mutations in the TTR gene. The disease is heterogeneous and clinical symptoms vary from cardiomyopathy to progressing sensorimotor polyneuropathy depending on TTR variant involved and the amyloid deposition site. Despite the fact that TTR-derived amyloid accumulates in different organs such as heart, kidney, eyes, and predominantly in the peripheral nerves of ATTR patients, the exact mechanism of the disease development is not understood. In contrast to the case of AD, it has been difficult to generate an animal model for ATTR in transgenic mice that would be useful in understanding TTR aggregation processes and the mechanisms of the associated toxicity as these mice did not develop any neuropathic phenotype besides amyloid deposits. Therefore, we created a disease-model in Drosophila due to its huge repertoire of genetic techniques and easy genotype – phenotype translation, as well as its success in modeling human neurodegeneration. We have generated transgenic flies that over-express the clinical amyloidogenic variant TTRL55P, the engineered variant TTR-A (TTRV14N ⁄ V16E), and the wild-type protein. All TTR variants were found in the secreted form in the hemolymph where misfolding occurred and depending on the pool of toxic species, the fate of the fly was decided. Within a few weeks, both mutants (but not the wild-type TTR) demonstrated a time-dependent aggregation of misfolded molecules in vivo. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability, and shortened life span. In contrast, expression of the wild-type TTR had no discernible effect on either longevity or fly behavior. In this work, we also addressed the correlation between TTR transgene dosage and thus, protein levels, with the severity of the phenotypes observed in TTR-A flies which developed a “dragged wing” phenotype. Remarkably, we established that degenerative changes such as damage to the retina strictly correlated with increased levels of mutated TTR but inversely with behavioral alterations and the dragged wing phenotype. We characterized formation of aggregates in the form of 20 nm spherules and amyloid filaments intracellularly in the thoracic adipose tissue and brain glia (both tissues that do not express the transgene). Moreover, we detected a fraction of neurotoxic TTR-A in the hemolymph of young but not old flies. We proposed that these animals counteract formation and persistence of toxic TTR-A species by removal from the circulation into intracellular compartments of glial and fat body cells and this is part of a mechanism that neutralizes the toxic effects of TTR. We validated the fly model for ATTR by applying a genetic screen during study of modifier genes. We found Serum amyloid P component (a product of the APCS gene) as a potent modifier of TTR amyloid-induced toxicity that was effective in preventing the apoptotic response in cell culture assay and capable of reducing the dragged wings when co-expressed in TTR-A flies. Finally, we optimized this fly model in order to screen for therapeutic compounds effective against ATTR. Feeding assays showed the effectiveness of several compounds among known native-state kinetic stabilizers of TTR against its aggregation. We described several early endpoints in this model, which can be used as a rapid and cost-effective method for optimizing concentrations and pre-screening of drug candidates. As the proof of principle, by feeding flies with increasing doses of diflunisal analogue (an FDA-approved Non-Steroidal Anti-Inflammatory Drug) a dose-dependent reduction of the dragged wings was observed.