Aldeídos de colesterol (Secosterol A e Secosterol B) têm sido detectados em amostras de cérebro humano e investigados em modelos de doenças neurodegenerativas como possíveis marcadores e intermediários do processo patológico. Estes oxisteróis constituem uma classe de eletrófilos derivados de lipídeos que podem modificar e induzir agregação de proteínas. A esclerose lateral amiotrófica (ELA) é um distúrbio neurodegenerativo associado ao acúmulo de agregados imunorreativos de superóxido dismutase (Cu, Zn-SOD, SOD1). O objetivo deste trabalho foi avaliar a presença de aldeídos de colesterol em ratos modelo ELA e sua capacidade de induzir a formação de agregados de SOD1 in vitro. Aldeídos de colesterol foram analisados no plasma, medula espinhal e córtex motor de ratos ELA. Uma quantidade elevada de Secosterol B foi detectada no córtex motor desses ratos em comparação com animais controle. Adicionalmente, os experimentos in vitro mostraram que Secosterol B e Secosterol A induziram a agregação da SOD1 em uma forma amiloidogênica que se liga à tioflavina T. Esta agregação não foi observada com o colesterol e os seus hidroperóxidos. Usando aldeídos de colesterol marcados com grupo alquinil e um ensaio de click chemistry, foi observado que os agregados de SOD1 estão ligados covalentemente aos aldeídos. A modificação covalente da proteína foi confirmada por análise de MALDI-TOF, que mostrou a adição de até cinco moléculas de aldeídos de colesterol à proteína por base de Schiff. Curiosamente, a análise comparativa com outros eletrófilos derivados de lipídeos (e.g. HHE e HNE) demonstrou que a agregação de SOD1 aumentou proporcionalmente à hidrofobicidade dos aldeídos, observando-se a maioragregação com aldeídos de colesterol. Os sítios de modificação da SOD1 foram caracterizados por nanoLC-MS/MS após digestão da proteína com tripsina, onde foram identificadas lisinas como o principal aminoácido modificado. Em geral, nossos dados mostram que a oxidação do colesterol que leva à produção de aldeídos de colesterol é aumentada no cérebro de ratos ELA e que os aldeídos altamente hidrofóbicos derivados de colesterol podem promover eficientemente modificação e agregação de SOD1. / Secosterol aldehydes (Secosterol B and Secosterol A) have been detected in human brain samples and investigated in models of neurodegenerative diseases as possible markers and intermediates of the pathological process. These oxysterols constitute a class of lipid-derived electrophiles that can modify and induce aggregation of proteins. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the accumulation of immunoreactive aggregates of superoxide dismutase (Cu, Zn-SOD, SOD1). The objective of this work is to evaluate the presence of secosterol aldehydes in ALS rats and their ability to induce formation of SOD1 aggregates in vitro. Secosterol aldehydes were analyzed in plasma, spinal cord and motor cortex of ALS rats. A higher amount of Secosterol B was detected in the motor cortex of these rats compared to control animals. In addition, in vitro experiments have shown that Secosterol B and Secosterol A induce aggregation of SOD1 into an amyloidogenic form that binds to thioflavin T. This aggregation was not apparent in incubations with cholesterol and its hydroperoxides. Using alkynyl-labeled secosterol aldehydes and a click chemistry assay, it was found that the SOD1 aggregates are covalently linked to the aldehydes. Covalent modification of the protein was confirmed by MALDI-TOF analysis, which showed the addition of up to five molecules of secosterol aldehydes to the protein by Schiff base formation. Interestingly, the comparative analysis with other lipid-derived electrophiles (e.g. HHE and HNE) demonstrated that the aggregation of SOD1 increased according to the hydrophobicity of the aldehydes. Compared to the other electrophiles, a higher SOD1 aggregation was observed with secosterol aldehydes. SOD1 modification sites were characterized by nanoLC-MS/MS afterprotein digestion with trypsin, revealing lysine as the major amino acid modified in these experiments. Collectively, our data show that cholesterol oxidation leads to the production of secosterol aldehydes, which are increased in the brain of ALS rats, and that these highly hydrophobic aldehydes can efficiently promote the modification and aggregation of SOD1.

Hepcidin is a well-known iron-regulatory hormone that plays a key role in maintaining peripheral iron homeostasis. The presence and wide-spread distribution of hepcidin in the brain suggests that this peptide may also be an important player in brain iron homeostasis. In this study, we tested the hypothesis that hepcidin exerts an important role in the regulation of brain iron content, which might benefit iron-associated NDs such as PD. We also examined the hypothesis that hepcidin could control iron transport processes via regulating iron transport proteins in the brain cells, thus maintaining brain iron homeostasis. / In conclusion, the results of the present study implied that hepcidin plays an important role in maintaining brain iron homeostasis. Hepcidin is beneficial for PD and this effect is related to its iron-regulatory effect via inhibiting iron accumulation in the substantia nigra. Hepcidin effectively controls iron uptake and release through regulating iron transport proteins expressions in the brain, which would contribute to brain iron homeostasis. Therefore, manipulation of hepcidin level in the brain has a potential to be developed into a novel preventive approach for the iron-associated NDs such as PD. / In the second part, we investigated the effect of hepcidin on the processes of iron uptake and release in the cultured brain cells including neurons, astrocytes and brain vascular endothelial cells (BVECs). The expressions of iron uptake proteins such as transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) as well as the iron exporter ferroportin 1 (Fpn1) were also observed. We found that hepcidin reduced both iron uptake and release via decreasing iron transport proteins expressions in these brain cells, which would contribute to its iron regulatory effect. Finally, we further explored the mechanisms underlying the regulatory effect of hepcidin on the iron transporters in the last part, and found that the action of hepcidin in reducing TfR1 expression is a direct and cAMP-PKA (Cyclic Adenosine 3', 5'-monophosphate/ Protein Kinase-A) pathway-dependent event. / Iron is a transition trace metal essential for mammalian cellular and tissue viability. It also plays important roles in the central nervous system (CNS), including embryonic brain development, myelination, and neurotransmitters synthesis. However, abnormal iron accumulation has been demonstrated in a number of neurodegenerative diseases (NDs) such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases (HD). Currently very little is known about the mechanisms involved in brain iron homeostasis and therefore it is not known why and how iron is abnormally increased in the brain. However, given the essential role that excess iron plays in the pathological processes in the NDs, to suppress the accumulated iron is expected to be an effective strategy to prevent and treat these NDs. / To investigate whether hepcidin could benefit iron-associated NDs including PD and whether this beneficial role is related to its iron-regulatory function in the brain, in the first part of study, we investigated the effects of hepcidin on the 6-hydroxydopamine (6-OHDA) in vitro and in vivo PD models. We found that in primary cultured mesencephalic (MES) neurons, hepcidin overexpression via adenovirus-hepcidin (Ad-hepcidin) infection prevented 6-OHDA-induced increase in cellular iron content and protected the MES neurons. In the 6-OHDA model of PD in vivo, overexpression of hepcidin in the substantia nigra via Ad-hepcidin intranigral injection significantly prevented iron accumulation and dopaminergic neurons loss in the pars compacta of substantia nigra (SNc). These effects were accompanied by a marked improvement in motor performance of the PD animals. These findings indicate that hepcidin could benefit iron-associated NDs such as PD and via its iron-regulatory role in the brain. / Du, Fang. / Adviser: Ya Ke. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The misfolding and aggregation of proteins is closely associated with more than fifty human disorders, including Alzheimer's and Parkinson's diseases, all of which are currently incurable and many represent a major threat to human life. The mechanism of protein aggregation is subject to extensive studies. The damaging effects associated with protein aggregation have been attributed to amyloidogenic species that are present during the misfolding process. In particular, oligomeric species are, however, intrinsically difficult to study as a consequence of their low abundance and highly heterogeneous nature. The first chapter of my thesis gives an introduction into the field of protein folding and misfolding with a focus on the study of protein aggregation, and toxic effects relevant to human disorders. The second chapter of my thesis describes the development of a methodology that enables the study of aggregate induced lipid bilayer permeability, possibly the most general mechanism of protein aggregate toxicity. Surface-tethered lipid vesicles functioning as optochemical probes sensitive to membrane integrity are imaged using total internal reflection microscopy. It is shown that oligomeric species of the 42-residue form of the Aβ peptide (Aβ42) are responsible for the membrane disruption. The methodology can be applied to the study of other proteins such as α-synuclein and tau, and the ability of antibodies and chaperones to counteract the aggregate induced lipid bilayer permeability can be assessed. Furthermore, lipid bilayer permeability induced by aggregates formed in human induced pluripotent stem cells can be studied. The third chapter presents a new approach for the measurement of protein aggregation kinetics by following the development of the lipid bilayer permeability over the course of the aggregation process of Aβ42. The aggregation kinetics can be modulated with molecular chaperones and pre-formed seed fibrils, which allows secondary nucleation to be identified as the process that drives the formation of species responsible for the lipid bilayer permeability. The fourth chapter describes the development of a three-pronged strategy to study the mechanism of α-synuclein amyloid formation. The aggregation is studied in the presence of lipid vesicles or pre-formed fibrils at neutral or acidic pH of the solution. The influence of single-point mutations on the aggregation of α-synuclein is described. Furthermore, the strategy is applied to the characterisation of the ability of antibodies and small molecules to inhibit the aggregation, and thus has the potential for the development of therapeutical agents. The work presented in the fifth chapter characterises the amyloid fibril populations formed by α-synuclein and mutational variants associated with familial Parkinson's disease. X-ray crystallography, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy and atomic force microscopy have all been applied to the analysis of these amyloid fibrils. Finally, the sixth chapter summarises the results described in this thesis and points out future opportunities in the context of fundamental and translational studies related to the research area of protein misfolding disorders.

De plus en plus de preuves montrent que le transport axonal rapide (FAT) joue un rôle crucial au cours des maladies neurodégénératives (NDs). La maladie de Huntington est une maladie neurodégénérative causée par une expansion anormale de polyglutamines dans la partie Nterminale de la protéine huntingtine (HTT) : une grande protéine d’échafaudage impliquée dans la régulation du transport. La présence de HTT mutante comme l’absence de la HTT induisent des défauts de transport chez les mammifères. Chez la Drosophile, la HTT mutante reproduit le phénotype observée chez les mammifères, cependant la fonction conservée de la HTT chez la Drosophile melanogaster (DmHTT) n’est pas encore clairement établie. Ici nous mettons en évidence que DmHTT s’associe aux vésicules, aux microtubules et intéragit avec la proteine dynéine. Dans les neurones corticaux de rat, DmHTT remplace partiellement la HTT de mammifère dans le transport axonal rapide, et les drosophiles invalidées pour la HTT montrent des défauts de transport axonal in vivo. Ces résultats suggèrent que la fonction de la HTT est conservée dans le modèle Drosophile.Le FAT est un processus qui requiert un apport constant d’énergie. Les mitochondries sont les principales sources de production d’ATP de la cellule. Cependant nous avons démontré que le FAT ne dépend non pas de cette source d’énergie là, contrairement à ce que l’on pensait, mais de l’ATP glycolytique produit par les vésicules. La dérégulation de GAPDH ou de PK, les deux enzymes glycolytiques productrices d’ATP, ralentit le transport vésiculaire. Néanmoins, l’invalidation de GAPDH n’affecte pas le transport mitochondrial. En outre, toutes les enzymes glycolytiques sont associées à des vésicules dynamiques et sont capables de produire leur propre ATP. Enfin nous montrons que l’ATP produit est suffisant pour assurer leur propre transport, prouvant l’autonomie énergétique des vésicules pour le transport. / Growing evidence support the idea that impairments in Fast Axonal Transport (FAT) play a crucial role in Neurodegenerative Diseases (NDs). Huntington’s Disease is neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-Terminal part of huntingtin (HTT), a large scaffold protein implicated in transport regulation. Both the presence of the mutated HTT as the loss of HTT leads to transport defects in mammals. In the fruit fly overexpression of the mutant HTT recapitulates the phenotype observed in mammals. However, it is still unclear whether HTT’s function is conserved in D. melanogaster. Here, we show that D. melanogaster HTT (DmHTT) associates with vesicles, microtubules, and interacts with dynein. In rat cortical neurons, DmHTT partially replaces mammalian HTT in fast axonal transport, and DmHTT KO flies show axonal transport defects in vivo. These results suggest that HTT function in transport is conserved in D. melanogaster.FAT is a process that requires a constant supply of energy. Mitochondria are the main producers of ATP in the cell. However, we have demonstrated that FAT does not depend on this source of energy, as previously thought, but it depends on glycolytic ATP produced on vesicles. Perturbing GAPDH or PK, the two ATP generating glycolytic enzymes, slows down vesicular transport. However, knocking down GAPDH does not affect mitochondrial transport. Furthermore, all of the glycolytic enzymes are associated with dynamic vesicles, and are capable of producing their own ATP. Finally, we show that this ATP production is sufficient to sustain their own transport, demonstrating the energetical autonomy of vesicles for transport.

Lam Wun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLDGEMENT --- p.iii / TABLE OF CONTENT --- p.iv / ABBREVIATIONS --- p.x / LISTS OF TABLES --- p.xi / LISTS OF FIGURES --- p.xii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegeneration and triplet repeat diseases --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Polyglutamine nuclear inclusions --- p.4 / Chapter 1.3.1 --- Kinetics of polyglutamine nuclear inclusion formation --- p.4 / Chapter 1.3.2 --- Roles of protein inclusions in neurodegeneration --- p.7 / Chapter 1.4 --- Polyglutamine pathogenic pathways --- p.8 / Chapter 1.4.1 --- Protein depletion theory --- p.9 / Chapter 1.4.2 --- Induction of apoptotic pathways --- p.13 / Chapter 1.5 --- Previous study on NI proteins --- p.14 / Chapter 1.6 --- Drosophila model for studying polyglutamine diseases --- p.15 / Chapter 1.6.1 --- Drosophila model for studying human diseases --- p.15 / Chapter 1.6.2 --- GAL4/UAS gene expression system --- p.15 / Chapter 1.6.3 --- Drosophila polyglutamine models --- p.17 / Chapter 1.7 --- Objectives of the study --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila genetics --- p.22 / Chapter 2.1.1 --- Drosophila culture --- p.22 / Chapter 2.1.2 --- GAL4/UAS gene expression system --- p.22 / Chapter 2.1.3 --- Eye phenotypic analysis --- p.25 / Chapter 2.1.4 --- Polyglutamine fly models --- p.25 / Chapter 2.1.5 --- Generation and characterization of GFP-polyglutamine transgenic fly models --- p.25 / Chapter 2.2 --- Proteomic identification of nuclear inclusion proteins --- p.26 / Chapter 2.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.26 / Chapter 2.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.27 / Chapter 2.2.2.1 --- Approach overview --- p.27 / Chapter 2.2.2.2 --- Sample preparation for two-dimensional gel electrophoresis --- p.27 / Chapter 2.2.2.3 --- Two-dimensional gel electrophoresis --- p.29 / Chapter 2.2.2.4 --- Polyacrylamide gel staining --- p.31 / Chapter 2.2.2.5 --- Computer analysis of 2D patterns --- p.31 / Chapter 2.2.2.6 --- In-gel trypsin digestion --- p.32 / Chapter 2.2.2.7 --- Mass spectrometric analysis --- p.33 / Chapter 2.2.3 --- Detection of NIs by flow cytometry --- p.34 / Chapter 2.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.34 / Chapter 2.3.1 --- Sample preparation for SDS-PAGE --- p.34 / Chapter 2.3.2 --- SDS-PAGE --- p.35 / Chapter 2.4 --- Immunodetection --- p.36 / Chapter 2.4.1 --- Electroblotting --- p.36 / Chapter 2.4.2 --- Western blotting --- p.36 / Chapter 2.4.3 --- Filter trap assay --- p.37 / Chapter 2.5 --- Sav antibody production --- p.38 / Chapter 2.5.1 --- Sav peptide synthesis --- p.38 / Chapter 2.5.2 --- Rabbit immunization --- p.38 / Chapter 2.6 --- Cryosectioning and immunostaining of adult fly heads --- p.39 / Chapter 2.7 --- Alcohol dehydrogenase assay --- p.40 / Chapter 2.8 --- Semi-quantitative reverse transcription- Polymerase Chain Reaction --- p.41 / Chapter 2.8.1 --- Total RNA preparation from fly heads --- p.41 / Chapter 2.8.2 --- Reverse transcription- Polymerase Chain Reaction (RT-PCR) --- p.41 / Chapter 2.9 --- Reagents and buffers --- p.42 / Chapter 3. --- RESULTS / Chapter 3.1 --- Transgenic polyglutamine fly models --- p.48 / Chapter 3.1.1 --- Characteristics of MJD polyglutamine fly model --- p.48 / Chapter 3.1.1.1 --- Overexpression of expanded truncated human MJD proteins in Drosophila causes eye degeneration --- p.49 / Chapter 3.1.1.2 --- Overexpression of expanded truncated human MJD proteins in Drosophila results in nuclear inclusion formation --- p.49 / Chapter 3.1.1.3 --- Formic acid dissolves fly polyglutamine nuclear inclusions --- p.51 / Chapter 3.1.1.3.1 --- Formic acid dissolves fly polyglutamine NIs as shown by Western blot analysis --- p.51 / Chapter 3.1.1.3.2 --- Formic acid dissolves fly polyglutamine NIs as shown by filter trap assay --- p.53 / Chapter 3.1.2 --- Summary --- p.55 / Chapter 3.2 --- Proteomic identification of nuclear inclusion (NI) proteins --- p.56 / Chapter 3.2.1 --- Proteomic identification of NI proteins by SDS-insolubility of NIs --- p.56 / Chapter 3.2.2 --- Proteomic identification of NI proteins by FA-solubility of NIs --- p.63 / Chapter 3.2.2.1 --- Two-dimensional gels showing differential protein spots as potential NI proteins --- p.63 / Chapter 3.2.2.2 --- NI protein candidates identified by the 2D approach --- p.75 / Chapter 3.2.3 --- Study of polyglutamine NI proteins by flow cytometry analysis --- p.90 / Chapter 3.2.3.1 --- Detection of fly polyglutamine NIs by flow cytometry --- p.90 / Chapter 3.2.3.2 --- Characterization of a new GFP-polyglutamine fly model --- p.92 / Chapter 3.3 --- Characterization of the nuclear inclusion protein candidates --- p.96 / Chapter 3.3.1 --- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) --- p.96 / Chapter 3.3.1.1 --- Confirmation of GAPDH as a NI protein --- p.97 / Chapter 3.3.1.2 --- Discussion --- p.97 / Chapter 3.3.2 --- Receptor of activated protein kinase C (RACK1) --- p.99 / Chapter 3.3.2.1 --- Confirmation of RACK1 as a NI protein --- p.99 / Chapter 3.3.2.1.1 --- Colocalization of RACK1 with NIs --- p.99 / Chapter 3.3.2.1.2 --- Formic Acid extracts RACK1 from NIs --- p.101 / Chapter 3.3.2.2 --- Reduction of soluble RACK1 protein level in polyglutamine fly --- p.101 / Chapter 3.3.2.2.1 --- Soluble RACK1 protein level reduced in polyglutamine fly --- p.101 / Chapter 3.3.2.2.2 --- RACK1 transcript level remains unchanged in polyglutamine fly --- p.103 / Chapter 3.3.2.3 --- Overexpression of RACK 1 partially suppresses polyglutamine degeneration --- p.105 / Chapter 3.3.2.4 --- Discussion --- p.107 / Chapter 3.3.3 --- Warts (Wts) --- p.111 / Chapter 3.3.3.1 --- Overexpression of Wts partially suppresses polyglutamine degeneration --- p.111 / Chapter 3.3.3.2 --- Wts mutant slightly enhances polyglutamine degeneration --- p.113 / Chapter 3.3.3.3 --- Genetic analysis of Warts pathway in polyglutamine pathogenesis --- p.113 / Chapter 3.3.3.3.1 --- Overexpression of Salvador partially suppresses polyglutamine degeneration --- p.116 / Chapter 3.3.3.3.2 --- Hpo mutant slightly enhances polyglutamine degeneration --- p.119 / Chapter 3.3.3.3.3 --- Overexpression of DIAP1 partially suppresses polyglutamine degeneration --- p.119 / Chapter 3.3.3.4 --- Discussion --- p.121 / Chapter 3.3.4 --- Alcohol dehydrogenase (Adh) --- p.122 / Chapter 3.3.4.1 --- Adh activity is reduced in polyglutamine flies --- p.122 / Chapter 3.3.4.2 --- Overexpression of Hsp70 partially restores the reduced Adh activity in polyglutamine flies --- p.122 / Chapter 3.3.4.3 --- Discussion --- p.125 / Chapter 3.3.5 --- Genetic analysis of other NI protein candidates --- p.127 / Chapter 3.3.5.1 --- Overexpression of CG7920 protein partially suppresses polyglutamine degeneration --- p.127 / Chapter 3.3.5.2 --- Pten dsRNA slightly enhances polyglutamine degeneration --- p.129 / Chapter 3.3.6 --- Summary --- p.131 / Chapter 4. --- DISSCUSSION / Chapter 4.1 --- Protein depletion theory --- p.133 / Chapter 4.2 --- Comparison of different approaches for identification of NI proteins --- p.134 / Chapter 4.3 --- Long-term significance --- p.136 / Chapter 4.4 --- Future studies --- p.137 / Chapter 4.4.1 --- Characterization of other NI protein candidates --- p.137 / Chapter 4.4.2 --- Study of NI proteins by an alternative approach --- p.137 / Chapter 4.4.3 --- Study of NI proteins using other polyglutamine fly models --- p.137 / Chapter 5. --- CONCLUSION --- p.139 / Chapter 6. --- REFERENCES --- p.140

Introdução: A doença por grãos argirofílicos (DGA) é uma tauopatia esporádica distinta, bastante frequente, com uma prevalência atingindo 31,3% em centenários, porém pouco reconhecida A manifestação clínica mais comum da DGA é de um comprometimento cognitivo de lenta evolução associado a uma alta frequência de sintomas psiquiátricos. O diagnóstico de DGA é possível somente através da análise do encéfalo post-mortem com os achados das três principais alterações patológicas: grãos argirofílicos, corpúsculos em embrião e pré-emaranhados neuronais. O presente estudo investigou as características demográficas, clínicas e neuropatológicas dos indivíduos com DGA e possíveis associações clínico-patológicas. Métodos: Foram estudados 983 casos (acima de 50 anos de idade) provenientes da amostra do Banco de Encéfalos do Grupo de Estudos em Envelhecimento Cerebral. A avaliação clínica e funcional foi realizada através de uma ampla entrevista semiestruturada respondida por um informante com contato próximo com o paciente. Os participantes foram estratificados conforme a presença de comprometimento cognitivo (de acordo com Escala de Avaliação Clinica da Demência) e, posteriormente, pela presença de DGA em quatro grupos: DGA com e sem comprometimento cognitivo e não-DGA com e sem comprometimento cognitivo. Análise descritiva foi realizada para dados socioeconômicos, genótipo de APOE e variáveis clínico-funcionais, neuropsiquiátricas e neuropatológicas na amostra DGA e em cada grupo. Foi utilizado um modelo de regressão logística multivariada para investigar as associações entre perfil cognitivo e sintomas neuropsiquiátricos com DGA. Resultados: DGA foi identificada em 150 indivíduos (15,1%). Idade avançada e baixo nível socioeconômico foram associados com DGA independente da presença de comprometimento cognitivo. A presença de DGA foi associada a uma redução de 60% na probabilidade de um escore >= 3.8 no Questionário do informante sobre o Declínio Cognitivo do Idoso (OR=0,40; IC de 95% 0,22-0,74; p=0,004). Adicionalmente, o subitem apetite do inventário neuropsiquiátrico foi associado à DGA em indivíduos cognitivamente normais (OR=1,85; IC de 95% 1,09-3,12; p=0,02). Conclusão: A DGA pode preservar a cognição em indivíduos com patologias neurodegenerativas associadas em particular nos casos com patologia tipo Alzheimer concomitante. A investigação dos mecanismos subjacentes a esse efeito pode auxiliar no desenvolvimento de novos tratamentos para a Doença de Alzheimer / Background: Argyrophilic grain disease (AGD) is an underrecognized, distinct, highly frequent sporadic tauopathy, with prevalence reaching 31.3% in centenarians. The most common presentation of AGD is a slowly progressive amnestic mild cognitive impairment, accompanied by high frequency of neuropsychiatric symptoms. AGD can only be diagnosed postmortem by the finding of its three main pathologic features: argyrophilic grains, oligodendrocytic coiled bodies and neuronal pretangles. The present study investigated demographic, clinical, and neuropathological profiles and analyzed clinicopathological associations. Methods: We studied 983 participants (over 50 years of age) from the Brain Bank of the Brazilian Aging Brain study group sample. Clinical and functional evaluation included demographics and a semi-structured interview covering various cognitive domains conducted with a knowledgeable informant. Participants were stratified by cognitive status (based on Clinical Dementia Rating scale), followed by the presence of AGD in four groups: AGD with and without cognitive impairmet, and non-AGD with and without cognitive impairment. Descriptive statistics were used for sociodemographic data, APOE genotypes, and the clinical, cognitive, neuropsychiatric, functional, and neuropathological variables in AGD samples and in each group. We used multivariate logistic regression models to investigate the association between the cognitive status and neuropsychiatric symptoms with AGD. Results: AGD was identified in 150 participants (15.1%). Older age and lower socioeconomic status were associated with AGD independent of cognitive status. Multivariate analyses revealed that AGD was associated with a 60% reduction in the odds of having an IQCODE >= 3.8 (OR = 0.40, 95% CI 0.22-0.74, p = 0.004) and that the NPI sub-item \"appetite and eating abnormalities\" was associated with AGD in controls (OR = 1.85, 95% CI 1.09-3.12, p = 0.02). Conclusion: AGD might preserve cognition in individuals with coexistent neurodegenerative pathologies, in particular those of the Alzheimer-type. Investigating whether the mechanisms underlying this effect could provide novel therapeutic approaches to the treatment of Alzheimer´s disease

A principal forma de tratamento da doença de Alzheimer é através de inibidores sintéticos de acetilcolinesterase. Os inibidores existentes no mercado além, de terem um custo elevado e apresentarem efeitos colaterais não previnem ou curam a doença de Alzheimer, apenas retardam o seu desenvolvimento. A baixa efetividade dos tratamentos existentes e a ausência de prognóstico positivo impulsionaram no mercado uma demanda por novas formas de tratamento e/ou prevenção da doença. Neste cenário, investigar a atividade anticolinesterásica em pimentas do reino e pimenta rosa, bem como seu potencial antioxidante e composição fitoquímica são maneiras de promover tanto o conhecimento sobre estas especiarias como beneficiar o panorama de saúde pública, seja no combate a doença de Alzheimer e/ou outras pandemias existentes. Com o objetivo de avaliar o potencial destas especiarias, extratos metanólicos de pimenta preta, verde, branca e rosa foram avaliados segundo o teor de fenólicos totais, atividade antioxidante pelo método DPPH, ABTS e FRAP e atividade anticolinesterásica. Posteriormente o perfil metabolômico dos grãos e extratos de pimenta foi analisado por cromatografia líquida acoplada à espectrometria de massa. As amostras foram ionizadas por -eletrospray? operando em modo negativo e positivo. O extrato de pimenta rosa apresentou o maior teor de compostos fenólicos totais e a melhor atividade antioxidante e anticolinesterásica em comparação aos demais extratos. Dentre as pimentas do reino, a pimenta verde destacou-se com os maiores teores de compostos fenólicos e atividade antioxidante pelo método DPPH e ABTS. Já a pimenta preta mostrou os melhores resultados para as análises de atividade anticolinesterásica e atividade antioxidante pelo método FRAP. Quanto ao perfil metabolômico as amostras de pimenta foram significativamente distintas entre si. Os espectros obtidos para as amostras de grãos de pimenta foram mais ricos em metabólitos em relação às amostras de extrato em ambos os modos operacionais. O processamento das pimentas do reino induziu modificações quantitativas e qualitativas sobre a composição fitoquímica deste condimento. A pimenta rosa não apresentou nenhum possível metabólito com alto potencial discriminatório, dentre os 15 íons investigados. Em modo operacional negativo, os metabólitos presentes no extrato de pimenta verde, exceto íon de m/z 329,2081, foram responsáveis por todas as contribuições mais significativas entre os perfis metabolômicos gerados. A partir dos resultados obtidos no presente trabalho é possível afirmar que a pimenta rosa apresenta o maior potencial na redução de risco da doença de Alzheimer, dentre as pimentas investigadas. / Synthetic inhibitors of cholinesterase are the main drugs used in the treatment for Alzheimer\'s disease and besides the high cost and side effects, the current drugs available in the market do not prevent or cure Alzheimer, only retard its development. The absence of positive prognosis and limited effectiveness of existing drugs enhance the demand for new forms of the disease\'s treatment and/or prevention. In this scenario, investigating Piper nigrum and Schinus terebinthifolius peppercorns for its acetylcholinesterase and antioxidant activity, as well as its phytochemical composition, are ways to promote both knowledge about these spices and the perspective of health, either in combat of Alzheimer\'s disease and/or other existing pandemics. In order to evaluate the potential of these spices, black, green, white and pink peppers methanolic extracts were evaluated according to the total phenolic content, antioxidant activity by DPPH, ABTS and FRAP assay and anticholinesterase activity. Subsequently the metabolomic profile of the extracts and peppercorns were analyzed by mass spectrometry coupled to liquid chromatography. Samples were ionized by electrospray operating in negative and positive modes. The pink pepper (Schinus terebinthifolius) extract showed the highest content of total phenolic compounds and the best antioxidant and anticholinesterase activity compared to the other extracts. Among Piper nigrum peppercorns, green pepper stood out as the highest levels of phenolic compounds and antioxidant activity by DPPH and ABTS methods. Black pepper obtained the best results for the analysis of acetylcholinesterase activity and antioxidant activity by FRAP method. Regarding the metabolomic profiles, pepper samples are significantly different from each other. The spectrum obtained for peppercorn\'s samples were richer in metabolites compared to extract\'s samples in both operating modes. The processing induced quantitative and qualitative changes on the phytochemical composition of the peppercorns. The pink pepper did not show any possible metabolite with high discriminatory potential, of the 15 ions investigated. In the negative operating mode, the metabolites present in the extracts of green pepper, excluding m/z 329.2081 ion, were responsible for all of the most significant contributions between metabolomic profiles generated. From the results obtained in this study it is possible to affirm that pink pepper has the greatest potential to reduce Alzheimer\'s disease risk, among peppers investigated.

Glycogen is the sole carbohydrate storage molecule found in mammalian cells and plays an important role in cellular metabolism in nearly all tissues, including the brain. Defects in glycogen metabolism underlie the glycogen storage diseases (GSDs), genetic disorders with variable clinical phenotypes depending on the mutation type and affected gene(s). Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy and a non-classical GSD. LD typically manifests in adolescence with tonic-clonic seizures, myoclonus, and a rapid, insidious progression. Patients experience increasingly severe and frequent epileptic episodes, loss of speech and muscular control, disinhibited dementia, and severe cognitive decline; death usually ensues in the second decade of life. LD, like one- third of all epilepsy disorders, is intractable and resistant to antiseizure drugs. A hallmark of LD is the accumulation of intracellular, insoluble carbohydrate aggregates known as Lafora bodies (LBs) in brain, muscle, and other tissues. LBs are a type of polyglucosan body, an insoluble aggregate of aberrant glycogen found in some GSDs and neurodegenerative disorders. Like most GSDs, LD is an autosomal recessive genetic disorder. Approximately 50% of LD patients carry mutations in the epilepsy, progressive myoclonus 2A (EPM2A) gene encoding laforin, a glycogen phosphatase. Remaining patients carry mutations in EPM2B, the gene that encodes malin, an E3 ubiquitin ligase. Laforin and malin play important roles in glycogen metabolism. In the absence of either enzyme, glycogen transforms into an insoluble, hyperphosphorylated and aberrantly branched polysaccharide reminiscent of plant starch. This abnormal polysaccharide precipitates to form LBs and has pathological consequences in the brain. Since a definitive LD diagnosis requires genetic testing, whole exome sequencing has been increasingly used to diagnose LD. As a result, numerous cases of more slowly progressing or late-onset LD have been discovered that are associated with missense mutations in EPM2A or EPM2B. Over 50 EPM2A missense mutations have been described. These mutations map to many regions of the laforin X-ray crystal structure, suggesting they produce a spectrum of effects on laforin function. In the present work, a biochemical pipeline was developed to characterize laforin patient mutations. The mutations fall into distinct classes with mild, moderate or severe effects on laforin function, providing a biochemical explanation for less severe forms of LD. LBs drive LD pathology. As a result, LBs and glycogen metabolism have become therapeutic targets. Since LBs are starch-like, and starch is degraded by amylases, these enzymes are potential therapeutics for reducing LB loads in vivo. However, amylases are normally secreted enzymes. Degradation of intracellular LBs requires a cell-penetrating delivery platform. Herein, an antibody-enzyme fusion (AEF) technology was developed to degrade LBs in vitro, in situ in cell culture, and in vivo in LD mouse models. AEFs are a now putative precision therapy for LD, potentially the first therapeutic to provide a significant clinical benefit. Prior to this work, LD was considered a homogenous disorder and treatments were only palliative. The data herein support a spectrum of clinical progression, a potential therapy for LD, and mechanistic insights into LD pathophysiology. This work illustrates how personalized medicine, both in diagnosis and treatment, can be achieved through basic biochemical approaches to human disease.

Friedreich's Ataxia (FA) is an autosomal-recessive, neurodegenerative disease characterized by progressive lower extremity muscle weakness and sensory loss, balance deficits, limb and gait ataxia, and dysarthria. FA is considered a sensory ataxia because the dorsal root ganglia and spinal cord dorsal columns are involved early in the disease, whereas the cerebellum is affected later. Balance deficits and gait ataxia are often evaluated clinically and in research using clinical rating scales. Recently, quantitative tools such as the Biodex Balance System SD and the GAITRite Walkway System have become available to objectively assess balance and gait, respectively. However, there are limited studies using instrumented measures to quantitatively assess and characterize balance and gait disturbances in FA, and longitudinal, quantitative analyses of both balance and gait have not been investigated in this patient cohort. The purpose of the present study was to characterize gait patterns of adults with FA and to identify changes in gait and balance over time using clinical rating scales and quantitative measures. Additionally, this study investigated the relationship between disease duration, clinical rating scale scores and objective measures of gait and balance. This study used a longitudinal research design to investigate changes in balance and gait in 8 adults with genetically confirmed FA and 8 healthy controls matched for gender, age, height, and weight. Subjects with FA were evaluated using the Berg Balance Scale (BBS), the Friedreich's Ataxia Rating Scale (FARS) and instrumented gait and balance measures at baseline, 6 months, 12 months and 24 months. Controls underwent the same tests at baseline and 12 months. Gait parameters were measured utilizing the GAITRite Walkway system with a focus on gait velocity, cadence, step and stride lengths, step and stride length variability and percent of the gait cycle in swing, stance and double limb support. Balance was assessed using the BBS and the Biodex Balance System; the latter included tests of postural stability and limits of stability. At baseline, there were significant differences in gait and balance parameters, BBS scores and FARS total scores between FA subjects and controls as determined using paired t-tests (p This is the first longitudinal study to demonstrate changes over time in gait and balance of adults with FA using both quantitative measures and clinical rating scales. This study provided a detailed characterization of the gait pattern and balance of adults with FA. The GAITRite Walkway system proved to be a sensitive measure, and able to detect subtle changes in gait parameters over time in adults with FA. In addition, the BBS was an appropriate and sensitive assessment to detect changes in static and dynamic balance in this patient cohort. Finally, results revealed a strong and consistent relationship between clinical rating scale scores, postural stability indices, limits of stability scores, and step and stride length variability in individuals with FA.

Multiple Sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). As part of this disorder the myelin sheath undergoes degeneration, leading to alterations in the conductivity of axons, and impaired function. The onset of the disease occurs in young adults and clinical pathology is characterised by varying severity. These include i) Relapsing Remitting MS (RR-MS), ii) Secondary Progressive MS (SP-MS) and iii) Primary Progressive MS (PP-MS). MS is more prevalent in women and accounts for more than two thirds of all MS sufferers. MS is considered to be a multifactorial disorder with both genetic and environmental components. The prevalence of MS is dependent on geographical localisation, with lower sunlight exposure linked to higher prevalence. Also, studies show an increased risk in close relatives, or in identical twins, indicating a significant genetic component to the disorder. There are a number of genes that may plausibly be involved in MS pathophysiology. These include myelin-related genes, such as the myelin basic protein (MBP), immune-related genes, such FC receptor and osteopontin, and heat shock proteins such as xb crystallin. These candidate genes have been implicated in a variety of ways but usually through immunological and/or genetic studies. One of the most consistent findings in recent years has been the association of disease with alterations in the specific major histocompatibility complex (MHC) localised to chromosome 6p21.3, and includes MHC I, II, III. Genome wide screens have permitted the identification of loci in the genome, which are associated with MS susceptibility. The number of genes involved in MS is unknown and several case-control association studies have been undertaken to reveal the involvement of potential candidate genes. In general terms, current research is aimed at determining allelic variation of candidate genes. Such genes have been implicated in MS because they reside within susceptible regions of the chromosome associated with MS or they have a plausible potential pathophysiological role in MS. Candidate loci investigated in this study, for association with MS susceptibility, include members of the nitric oxide synthase family of metabolic proteins (inducible NOS, iNOS/NOS2A and neuronal NOS, nNOS), methylenetetrahydrofolate reductase (MTHFR), catechol-O-methyl transferase (COMT), and vitamin D receptor (VDR). The MS population used in all studies consisted of over 100 MS cases and gender, age and ethnicity matched controls. In our study of inducible and neuronal NOS genes, PCR based assays were developed to amplify a region of both promoters that contained known microsatellite variation. Supporting phyisological data suggests that the neuroinflammatory aspects of MS are associated with aberrant NO production, which may be due to aberrant regulation of NOS activity. Specific amplified products were identified by fluorescent capillary electrophoresis and allele frequencies were statistically compared using chi-squared analysis. In the nNOS and iNOS study, no association was identified with allele frequency variation and MS susceptibility (nNOS: ?2=5.63, P=0.962; iNOS: ?2=3.4; P=0.082). Similarly, no differences in allele frequencies were observed for gender or clinical course for both markers (Pvalue greater than 0.05). In short, results from this study indicate that the NOS promoter variations studied do not play a significant role in determining susceptibility to MS in the tested population. The COMT and MTHFR genes are localised at 22q12-13 and 1p36.3 respectively, regions of the genome that have been found to be positively associated with MS susceptibility. In our research, we set out to examine the G158A change in the 4th exon of the COMT gene. This functional mutation leads to an amino acid change (valine to methionine) that is directly associated with changes in the activity of COMT. The MTHFR enzyme plays a role in folate metabolism, and can be implicated in the turnover of homocysteine. Previous investigations have shown that high levels of homocysteine are encountered in MS patients, where it is also linked to demyelination in the CNS. In our study the aim was to examine the C677T variation (alanine to valine amino acid change) in the exon 4 coding region of the MTHFR gene and the G158A variation in the COMT gene. Restriction fragment length polymorphism (RFLP) analysis and gel electrophoresis was used to identify specific alleles for both COMT and MTHFR. However, as with the NOS study, no specific association was identified between MS susceptibility and variation for either of the tested COMT or MTHFR (Pvalue greater than 0.05) variants. In a final genomic investigation of the MS population, three variations in the VDR gene were analysed for association with MS susceptibility and pathology. Using RFLP analysis, three VDR variants were investigated with genotypes detected using the Taq I, Apa I and Fok I restriction enzymes. In contrast to previous genotypic analyses, this study did show a positive association, specifically between the functional variation in exon 9 of the VDR gene and MS (Taq I, 2= 7.22, P= 0.0072). Interestingly, the Apa I variant of VDR was also found to be associated with MS ( 2=4.2, P=0.04). The Taq I and Apa I variants were also found to be in very strong and significant linkage disequilibrium (D'=0.96, Pvalue less than 0.0001) and their associations were more prominent with the progressive forms of MS (SP-MS and PP-MS). In addition to genotypic analysis of a clinical population, additional research was undertaken to identify novel targets for MS susceptibility studies. Global gene expression analysis was undertaken using comparative subtractive fluorescent microarray technology to examine differences in gene activity (expression) in age and sex matched MS plaque tissue and anatomically matched normal white matter (NWM). MS plaques were obtained post mortem from MS sufferers with no drug history in the last two months before death and matched anatomically to healthy white matter from donors with no previous neurological disorders. Target arrays consisted of 5000 cDNAs and analysis was conducted using the Affymetrix 428 scanner. In this way, 139 genes were shown to be differentially regulated in MS plaque tissue compared to NWM. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (Pvalue less than 0.0001, a=0.73); while 70 transcripts were uniquely differentially expressed ( 1.5-fold) in either acute or chronic active lesions. To validate the gene expression profile results, quantitative real time reverse transcriptase (RT) PCR (Q-PCR) analysis was performed. 12 genes were selected because they were shown to be differentially expressed by array analysis in this study, or because of their involvement in MS pathology. These included transferrin (TF), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), glutathione S-transferase pi (GSTP1), crystallin, alpha-B (CRYAB), phosphomannomutase 1 (PMM1), tubulin beta-5 (TBB5), inositol 1,4,5-trisphosphate 3-kinase B (ITPKB), calpain 1 (CAPNS1), osteopontin (SPP1 or OPN), as well as the signal transducer and activator of transcription 1 (STAT1) and protein inhibitor of activated STAT1 (PIAS1). Both absolute (copy number) and comparative differences in the relative levels of expression in MS lesions and NWM were determined for each gene. The results from this study revealed a significant correlation of real time PCR results with the microarray data, while a significant correlation was also found between comparative and absolute determinations of fold. As with the results of array analysis, a significant difference in gene expression patterning was identified between chronic active and acute plaque pathologies. For example, a up to 50-fold increase in SPP1 and ITPKB levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P less than 0.0.1, unpaired t-Test). Of particular note, gamma-amino butyric acid receptor ?2 (GABG2), integrin ?5 (ITGB5), complement component 4B (C4B), parathyroid hormone receptor 1 (PTHR1) were found up-regulated in MS and glial derived neurotropic factor ?2 (GDNFA2), insulin receptor (INSR), thyroid hormone receptor ZAKI4 (ZAKI4) were found down-regulated in MS. Data also revealed a decreased expression of the immune related genes STAT1 and PIAS1 in acute plaques. In conclusion, this research used both genomic analysis and technologies in gene expression to investigate both known and novel markers of MS pathology and susceptibility. The study developed tools that may be used for further investigation of clinical pathology in MS and have provided interesting initial expression data to further investigate the genes that play a role in MS development and progression.