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<p>Advances in modern medicine have helped to prolong human life. These advancements coupled with an ever-increasing population means that diseases associated with aging will become more prevalent in the coming years. As such, it is critical to understand the pathogenesis of disease where aging is the main risk factor. While not widely known, age is in fact a large risk factor in development of obesity and metabolic syndrome. More widely known and discussed are the neurodegenerative diseases that occur late in life. While age as a risk factor is a common point between these types of pathology, there are other similarities, such as the interaction between lipid metabolism and mitochondrial health. </p><p>To study the overlap between obesity and neurodegeneration, we investigated two pathways that regulate both. First, we find that loss of cytoplasmic deacetylase HDAC6 leads to aberrant accumulation of lipid in vitro and in vivo. HDAC6 knock-out (KO) mice gain more weight than WT counterparts after a high-fat diet regimen. Additionally, the intermediary metabolism of cells lacking HDAC6 is disrupted as they increase glucose uptake while downregulating fatty acid oxidation. HDAC6 not only plays a role in lipid metabolism, but regulates mitochondrial dynamics. Upon glucose-withdrawal, HDAC6 KO cells fail to elongate their mitochondria and display increased levels of mitochondrial toxic by-products. Therefore, HDAC6 has critical roles in lipid homeostasis and mitochondrial health. </p><p>The other pathway we investigated is critical in neurodegenerative disease, Parkinson's disease. Parkin, an E3 ubiquitin ligase, flags damaged mitochondria for destruction so they do not poison the other functional organelles. We found that Parkin promotes lipid remodeling at the surface of the mitochondria. Phosphatidic acid (PA) accumulates shortly after mitochondrial damage while diacylglycerol (DAG) appears several hours later. This lipid accumulation is dependent upon Parkin's translocation and E3 ligase activity. Additionally, we found that lipin-1, a PA phosphatase, and endophilin B1 (EndoB1) are critical for DAG accumulation and effective mitochondrial clearance. </p><p>Through this work, we show that two proteins critical in quality control mechanisms also play significant roles in energy homeostasis. We aim to highlight this overlap and posit that common diseases of aging, though presenting differently, might have disruptions in the same basic process.</p> / Dissertation

Title: The role of nitric oxide in the pathophysiology of neurodegenerative diseases Objectives: The main objective of this thesis is to evaluate the effect of nitric oxide on the formation and development of neurodegenerative diseases. Another objective was to determinate, whether NO affects by its impact processes involved in apoptosis in the CNS. Methods: The thesis is prepared in the form of research, drawing from available relevant resources. Results: Nitric oxide is widely applied in the pathophysiology of selected neurodegenerative diseases, either directly or through other reactive nitrogen and oxygen. It also affects other factors that are involved in apoptosis in the CNS. Keywords: Nitric oxide, NMDA receptors, neurodegenerative diseases, excitotoxicity, apoptosis

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As epilepsias acometem entre 1-2% da população mundial. De um modo geral, de todas as epilepsias quase um terço deste total de pacientes apresenta a síndrome epiléptica conhecida como Epilepsia de Lobo Temporal (ELT), a qual se instala geralmente após um insulto inicial ou em decorrência de outras patologias como, por exemplo, trauma ou tumor, e parece ser decorrente de anormalidades intrínsecas do lobo temporal tais como, amígdala, hipocampo e córtex piriforme. Depois de um período de latência variado, promove o surgimento de crises convulsivas. Dentre os pacientes que apresentam ELT, cerca de 20 a 30% deles apresentam resistência ao tratamento farmacológico. Para melhor estudar os processos plásticos envolvidos no processo de epileptogênese ocorridos após a instalação do insulto inicial que levam ao aparecimento de crises recorrentes espontâneas, ratos Wistar foram eletricamente estimulados na amígdala para indução de Status Epilepticus (SE). Foram feitas histoquímicas e immunohistoquímica para marcar neurônios ativados (c-Fos+), novos neurônios (Doublecortin DCX+) e em degeneração (FluoroJade C - FJC+) após as crises. Após a indução do SE observamos que quanto mais graves as crises, maior o número de áreas ativadas (c-Fos+) e maior número de neurônios em degeneração (FJC+). Além disso, não houve associação direta entre as áreas cerebrais ativadas e grau de neurodegeneração, nem associação entre gravidade do SE e intensidade de neurogênese (DCX). A segunda fase deste projeto, executada na University of Cincinnati, refere-se ao estudo do impacto do SE, induzido por pilocarpina (PILO) sistêmica, sobre a neurogênese hipocampal. Utilizando a injeção de BrdU, para marcar o dia do nascimento de novos neurônios granulares, em camundongos Thy1-GFP foram submetidos ao SE por PILO. Foram analisadas a plasticidade dendrítica de neurônios granulares em fase de maturação (imaturas, 1 semana) e maduras (8 semanas). As células imaturas sofreram drásticas modificações na sua morfologia e na densidade dendrítica. Por outro lado, as células maturas não sofreram alterações morfológicas na árvore dendrítica, mas apresentaram uma intensa redução na densidade dos espinhos dendríticos, mostrando assim que as células imaturas estão mais suceptíveis ao impacto das crises epilépticas. / The epilepsies affect between 1-2% of the world. In general, all epilepsies almost a third of total patients had an epilepsy syndrome known as temporal lobe epilepsy (TLE), which usually settles after the initial insult or due to other pathologies such as, for example, trauma or tumor, and seems to be due to intrinsic abnormalities such as temporal lobe, amygdala, hippocampus and piriform cortex. After latency period varied, promotes the emergence of seizures. Among the patients with TLE, about 20 to 30% of them are resistant to pharmacological treatment. To better study the processes involved in plastic epileptogenesis occurred after the installation of the initial insult leading to the appearance of spontaneous recurrent seizures, rats were electrically stimulated in the amygdala to induce status epilepticus (SE). Histochemical and immunohistochemistry were done to mark neurons activated (c-Fos +), newborn neurons (Doublecortin - DCX+) and degenerating (FluoroJade C - FJC+) after the crisis. After SE induction observed that the more serious crises, the greater the number of activated areas (c-Fos+) and greater number of degenerating neurons (FJC+). In addition, there was no direct association between the brain areas activated and the degree of neurodegeneration, or association between the severity and intensity of the SE of neurogenesis (DCX+). The second phase of this project, performed at the University of Cincinnati, refers to study the impact of SE induced by pilocarpine (Pilo) system on hippocampal neurogenesis. Using the injection of BrdU, to label the daybirth of new granule neurons in Thy1-GFP mice subjected to SE. We analyzed the dendritic plasticity of granule neurons undergoing maturation (immature, 1 week) and mature (8 weeks). The immature cells have undergone drastic changes in their dendritic morphology and density. On the other hand, the mature cells did not undergo morphological changes in dendritic tree but showed a marked decrease in the density of dendritic spines, thus showing that immature cells are more susceptible to the impact of epileptic seizures.

Thesis advisor: Thomas N. Seyfried / Gangliosides are glycosphingolipids (GSLs) containing sialic acids that play numerous roles in neuronal maturation, apoptotic signaling, angiogenesis, and cell surface receptor activity. The GM1 and GM2 gangliosidoses are a series of autosomal recessive lysosomal storage disorders (LSDs) characterized by an inability to degrade these lipid molecules. GM1 gangliosidosis is caused by a mutation in the lysosomal hydrolase β-galactosidase, resulting in neuronal storage of ganglioside GM1 and asialo GA1. Tay-Sachs (TS) and Sandhoff Disease (SD) are GM2 gangliosidoses caused by mutations in either the α or β subunits, respectively, of the heterodimeric protein β- hexosaminidase A, resulting in the storage of ganglioside GM2 and asialo GA2. The accumulation of excess ganglioside in the central nervous system leads to abnormal intracellular vacuoles, neuronal loss, demyelination, ataxia, dementia, and premature death. In my studies, I have shown that accumulation of GM1 ganglioside may not coincide with secondary storage of cholesterol, by providing evidence that cholesterol-binding fluorescent molecule filipin reacted to GM1 ganglioside in the absence of cholesterol. In an effort to combat the early-onset gangliosidoses, I have explored the effects of combining Neural Stem Cells (NSCs) with Substrate Reduction Therapy (SRT) in juvenile Sandhoff mice. The analysis showed that SRT was more effective than NSCs in reducing stored GM2 and GA2 in young mice, and no synergy was observed. In adult GM1 gangliosidosis, Tay- Sachs, and Sandhoff mice, Adeno-Associated Viral (AAV) vector gene therapy was used to restore therapeutic levels of wild-type enzyme to the CNS. AAV therapy corrected ganglioside storage and ameliorated myelin-associated lipid loss in all tissues assayed, increasing motor performance and life in effected animals. Lastly, AAV therapy was also successful in a feline model of Sandhoff disease. These results in juvenile and adult model systems point the way towards multiple effective clinical therapies in the near future. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Alzheimer’s disease (AD) is a severe age-related neurodegenerative disorder and is the most common form of dementia. Although the pathogenesis of AD remains unknown, the deterioration of the cerebrovascular system constitutes a risk factor associated with the development of the disease. Notably, brain hypoperfusion, a feature of healthy ageing brain and AD, occurs prior to the onset of cognitive decline in AD and correlates with the severity of dementia. Although there is a clear link between hypoperfusion and cognitive alterations in AD, a causal relationship remains to be established. It was hypothesised that chronic cerebral hypoperfusion leads to the accumulation of parenchymal and vascular amyloid-β (Aβ), triggering the development of vascular lesion (microinfarcts (MIs) and haemorrhages) and altering the neurovascular unit (NVU) integrity. Second to this, it was hypothesised that reductions in Aβ levels by immunotherapy targeted to amyloid in young mice, reduce amyloid levels, and prevent vascular lesions improving cognitive performance. Three studies were conducted to test these hypotheses. In the first study, the aim was to characterise age-dependent changes in amyloidrelated pathology in a transgenic mouse model (Tg-SwDI). The temporal amyloid precursor protein (APP) expression, accumulation of parenchymal and cerebrovascular Aβ and Aβ-related microglial and astrocytic activation in the cortex, hippocampus and thalamus of the Tg-SwDI mice at 3, 6 and 9 months of age was compared to wild-type controls. Significantly higher APP expression (p < 0.05), as well as Aβ aggregation (p < 0.001) as the animals aged was found in the Tg-SwDI mice in all the brain regions analysed, which was accompanied by extensive and progressive activation of microglial (p < 0.001) and astrocytic (p < 0.01) cells. These data provided a basis to design the next studies, as it was planned to induce hypoperfusion in these mice before significant Aβ deposition occurs. In the second study, the aim was to investigate the effect of hypoperfusion on Aβ dynamics and subsequently, to study the contribution of hypoperfusion and Aβ pathology to the development of MIs and haemorrhages, and to the potential alteration of astrocyte and tight junction (TJ) integrity. To address this, mild chronic cerebral hypoperfusion was induced in Tg-SwDI and wild-type mice by bilateral common carotid stenosis for 1 and 3 months. A significant increase in soluble Aβ40/42 levels was initially found after 1 month of hypoperfusion in the parenchyma (Aβ40, p = 0.0239; Aβ42 p = 0.0198) in parallel with elevated APP levels and APP proteolytic cleavage products (p < 0.05). Thereafter, following 3 months, a significant increase in insoluble Aβ40/42 levels was determined in the parenchyma (Aβ40, p = 0.0024; Aβ42 p = 0.008) and vasculature (Aβ40, p = 0.0046; Aβ42 p = 0.0118) of Tg-SwDI mice. There was no change in the levels of Aβ co-localised to vessels following 1 month of hypoperfusion; however Aβ levels were significantly increased in cerebral vessels after 3 months (p = 0.0483). The proportion of Aβ containing vessels was significantly higher in the small vessels of the hypoperfused animals compared to sham mice (p < 0.05). MIs associated with microglial proliferation were present in the Tg-SwDI mice and the burden was exacerbated by hypoperfusion at 1 and 3 months (p < 0.05). Significantly higher levels of NADPH Oxidase-2 (NOX2) were found in the transgenic mice compared to the wild-type controls at both time-points analysed (p < 0.05), and this was exacerbated after 1 month of hypoperfusion in the Tg-SwDI mice (p < 0.05). There was a positive correlation between NOX2 and soluble parenchymal Aβ levels (r = 0.6643, p = 0.0019). A minimal effect on the development of haemorrhages at these time-points was observed. In parallel to this, astrocyte activation was significantly higher in the Tg-SwDI mice compared to the wild-type controls at both time-points studied (p < 0.05); however, no effect of hypoperfusion was observed. Also, significantly higher levels of aquaporin-4 (AQP4) in the Tg-SwDI mice compared to the wild-type controls following 1 month of hypoperfusion were found (p < 0.001). There was a positive correlation between AQP4 and soluble parenchymal Aβ levels (r = 0.4735, p = 0.0095). Claudin-5 levels were significantly higher in the Tg-SwDI mice compared to the wild-type controls at both time-points analysed (p < 0.0001), and this was exacerbated following 1 month of hypoperfusion in the transgenic model (p < 0.05). A positive correlation between claudin-5 and vascular Aβ levels was observed (r = 0.6113, p = 0.0004). Together, these data suggest a synergistic contribution of amyloid and hypoperfusion pathologies to the tissue damage and implicate a role of oxidative stress and inflammation. In the third study, the aim was to determine the effects of passive amyloid immunisation on Aβ levels, development of MIs and haemorrhages and behavioural performance in the Tg-SwDI mice. To address this, the mice underwent weekly intraperitoneal injections with either 3D6 or 10D5 antibodies during 3 months. Although there were no significant changes between control and 10D5/3D6 treated mice in amyloid levels, appearance of MIs and cognitive performance, it was noted that there was a trend towards a reduction in amyloid levels and MI area in the 10D5/3D6 treated mice compared to the control animals. Furthermore, there was no evidence of microhaemorrhages in response to the immunisation. These results demonstrate that Aβ immunotherapy with the antibodies 3D6 and 10D5 may potentially decrease parenchymal and vascular amyloid accumulation, reducing the appearance of MIs and notably without triggering the development of microhaemorrhages. Collectively, the findings presented in the current thesis demonstrate that chronic cerebral hypoperfusion increases parenchymal and vascular Aβ levels and point towards a mechanism in which the cascade of events including inflammation and oxidative stress, triggered synergistically by hypoperfusion and Aβ, resulted in the widespread development of MIs and NVU changes which may further induce the alteration of cognition networks. A mixed therapy, aimed at improving cerebrovascular health and targeting the accumulation of Aβ, represents a promising strategy to prevent neurodegenerative processes and further cognitive decline in AD.

The aim of my thesis was to develop and characterise PrP transgenic Drosophila melanogaster of various genotypes to study the process of prion-induced neurodegeneration in this model. Prion diseases are caused by the occurrence of an abnormally-folded form of PrP (PrPSc) protein that arises either from the environment as an acquired disease, from mutation in the PrP-coding gene as a genetic disease or sporadically from causes unknown. The PrPSc then recruits PrPC, the normal form of PrP, that is ubiquitously present in the mammalian CNS and triggers neurotoxicity and neurodegeneration that is transmissible between individuals of the same or even different species. All prion diseases are currently incurable, fatal and the mechanism of prion-induced neurodegeneration remains to be discovered. In this thesis, Drosophila transgenic for ovine (chromosome 3 and dual PrP transgenic flies), hamster, humanised murine, human and cervid PrP were characterised for expression and biochemical properties. The ultimate goal of my thesis was investigation of cell-to-cell spread of misfolded PrP in Drosophila CNS. To achieve this, a mutant form of PrP that is thought to misfold was co-expressed with the normal form PrPC that served as a substrate in the same dual PrP-transgenic fly. The process was modelled using hamster, humanised murine or ovine PrP transgenes that carry human mutations associated with the spontaneous onset of transmissible neurodegeneration in the natural host. Various approaches towards independent spatial expression of PrP in Drosophila were exploited here in both single and dual PrP expressing flies. Moreover, the ability to initiate misfolding and the impact of this on the fly phenotype was investigated. Both apparent misfolding and phenotypic changes were seen in different fly models suggesting the models were successful. To this extent, PrP transgenic Drosophila were developed to allow for relatively rapid modelling of mammalian prion disease in this invertebrate organism.

Introdução: A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum. Os sintomas motores são decorrentes da morte de neurônios dopaminérgicos da Substância Nigra mesencefálica e por inclusões intracitoplasmáticas de ?-sinucleína, os corpúsculos de Lewy (CL). A doença pode ser o resultado de fatores ambientais agindo sobre um indivíduo geneticamente susceptível. O objetivo desse estudo foi verificar a frequência de mutações no gene PARK8/LRRK2 em uma amostra de pacientes brasileiros portadores de DP e descrever as principais correlações clínicas encontradas nos pacientes com mutações. Metodologia: Estudo transversal baseado no protocolo padronizado pelo projeto LARGE-PD (Latin American Research Consortium on The Genetics of PD) aplicado em 282 pacientes com DP recrutados de ambulatórios especializados em Distúrbios do Movimento do Hospital das Clínicas de Ribeirão Preto/USP e do Hospital São Paulo/UNIFESP, entre os anos de 2007 e 2014. O material genético colhido foi enviado para Seattle, com análise genética realizada no laboratório do Dr. Cyrus Zabetian da Universidade de Washington. Resultados: Realizado pesquisa genética para o LRRK2 em 229 pacientes de 282 pacientes que preencheram o protocolo. Quatro (1,74%) pacientes foram positivos para a mutação. Nos casos de inicio precoce, a frequência foi de apenas um caso (2,43% - 1/41). Três pacientes tinham história familiar positiva para DP (3,7% - 3/81). A idade de inicio dos sintomas variou entre 38 e 55 anos. A mutação G2019S esteve presente em 1,31% (3/229). Foi encontrado também um caso de mutação para R1441C. Conclusões: O LRRK2 se mostrou um importante gene correlacionado a DP, tendo como principal mutação a G2019S. O início dos sintomas variou entre 38 e 55 anos, sempre unilateral, com boa resposta a Levodopa. / Introduction: Parkinson\'s disease (PD) is the second most common neurodegenerative disease. Motor symptoms are due to the death of dopaminergic neurons in the midbrain Substance Nigra and intracytoplasmic inclusions known as Lewy bodies (CL), rich in a protein called ?-synuclein. The disease can be the result of environmental factors acting on an individual genetically susceptible, multifactorial etiology. The aim of this study was to determine the frequency of mutations in the gene PARK8 / LRRK2 in a sample of brazilian patients with PD and describe the main clinical correlations in patients with mutations. Methodology: This is a crosssectional study based on a standardized protocol for LARGE-PD project (Latin American Research Consortium on The Genetics of PD) applied in 282 patients with PD recruited from specialized clinics in Movement Disorders seen at Hospital das Clínicas de Ribeirão Preto/USP and Hospital São Paulo/UNIFESP, between the years 2007 and 2014. The genetic material was sent to Seattle, and genetic analysis was performed in the laboratory of Dr. Cyrus Zabetian at the University of Washington. Results: Realized genetic research for LRRK2 in 229 patients of 282 patients who met the LARGE-PD protocol. Observed four (1,74%) patients positive for the mutation. In cases of early-onset, the frequency was only one case (2,43% - 1/41). Three patients had a family history of PD (3,7% - 3/81). The age of onset of symptoms in patients with mutations varied between 38 and 55 years. A total of PD patients with the DNA analyzed, G2019S was present in 1,31% (3/229). It was also found one case to mutation R1441C. Conclusions: The LRRK2 had great influence gene correlated with PD, the main mutation G2019S. The onset of symptoms varied between 38 and 55 years, always one-sided, with good response to levodopa.

Parkinson's disease is a progressive neurodegenerative disorder of the central nervous system, manifesting with both motor and non-motor symptoms. Autosomal recessive mutations in the FBXO7 gene have been identified to cause a rapidly progressing early-onset form of PD. Canonically, FBXO7 functions as a substrate-recruiting subunit of the SCF-type E3 ubiquitin ligase. However, it also has a variety of other atypical functions, such as cell cycle regulation, proteasome regulation, and mitophagy. The overall aim of this research was to characterise the functional role of FBXO7 in various in vitro and in vivo PD models. The models examined included FBXO7 shRNA knockdown SH-SY5Y cell lines, FBXO7 CRISPR knockout SH-SY5Y cell lines, primary patient fibroblasts with a FBXO7 mutation, and MEFs and tissues from a Fbxo7 KO mouse. My analysis of fibroblasts from a patient without FBXO7 expression revealed several interesting phenotypes. Briefly, the patient fibroblasts proliferated slower due to increased apoptosis and lower CDK6 and cyclin D1 expression, which led to fewer cells progressing through the G1 phase of the cell cycle. My experiments showed that these cells also had mitochondrial respiration defects, exhibiting lower basal respiration, ATP production, maximal respiration and spare capacity, in addition to complex I, III and IV deficiencies. Patient fibroblasts also had significantly lower levels of 12S and 16S ribosomal mRNA transcripts, which are necessary for the translation of mitochondrially encoded subunits of complexes I, III, and IV. Similar phenotypes were also observed in MEFs from a Fbxo7 KO mouse model, indicating conservation between human and mouse FBXO7 in regulating mitochondria, cell death and proliferation. In a tissue-specific KO mouse model of PD, where FBXO7 expression was ablated in the dopaminergic neurons, I analysed proteins regulated by FBXO7 which might be responsible for cell loss in the substantia nigra. I discovered that RPL23, a regulator of MDM2, was ubiquitinated by SCFFbxo7 using K48 chain linkages, promoting its degradation by the proteasome. This suggests that misregulation of the MDM2:p53 axis may underlie the cell loss observed in this conditional Fbxo7 KO mouse model. In conclusion, these results elaborate on the role of FBXO7 in mitochondrial biology, and identify a new ubiquitination substrate of FBXO7 in a mouse model of PD. It is hoped that by elucidating the potential pathogenic mechanisms of FBXO7 in rare familial forms of the disease, it will be possible to translate findings to the more prevalent sporadic forms of Parkinson's disease as well.