Alzheimer’s disease has been described long ago, yet the illness is yet to be fully understood. Though it is true that research and technological advancements have brought us closer to understanding the disease, a truly effective pharmacological cure has not been discovered. With no permanent cure to rely on, AD patients and their caregivers still go through profound struggles in navigating through life with the disease. In this thesis, current literature on the non-pharmacological interventions is presented and discusses the various options that can provide the greatest relief and reap the most health benefits for patients. In total, four different non-drug treatments come into discussion - exercise, music, diet and cognitive interventions. In terms of exercise, research suggests that anaerobic work may be more beneficial than aerobic exercises in preventing the development or progression of mild cognitive dementia and Alzheimer’s disease. This is mostly due to the fact that anaerobic exercises can shift APP processing away from the non-amyloidogenic pathway and increase BDNF levels to offer improved neural protection. Music therapy intervention is evaluated next. This unique treatment is highly valued due to its beneficial effects on AD patients’ emotional well-being. Music therapy can take the forms of singing in groups or as an individual, and it can also incorporate dancing. Not only does music promote neuroplasticity and neurogenesis, but it also alleviates mood, boosts confidence and strengthens will. Diet is another significant component that can have an incredible impact on the AD patients’ wellbeing. Research reveals that diets high in saturated fatty acid should be avoided. On the other hand, diets mirroring the Mediterranean diet, including polyunsaturated fatty acids along with high amounts of vitamin C and folic acid should be readily consumed. Moreover, spices and herbs such as capsaicin should be used in a limited manner to decrease risk for AD. Finally, cognitive therapy is still a popular method for treating mild cognitive impairment and AD. Though cognitive improvement appears to be more modest, some psychostimulation programs combined with pharmacological treatments can play an influential role in achieving cognitive stability. Further research is needed in upgrading the current non-pharmacological interventions with an emphasis on the four treatments. These are available at an affordable cost and can be easily incorporated into the lifestyles of Alzheimer’s patients.

Alzheimer’s disease (AD), one of the major causes of disability and mortality in Western societies, is a progressive age-related neurodegenerative disorder. Increasing evidence suggests the etiology of AD may involve disruptions of zinc (Zn) homeostasis. We hypothesize that disruption of Zn homeostasis leads to alterations of Zn transporter (ZnT) proteins, resulting in increased production of neurotoxic amyloid beta (Aβ) peptide in AD brain. To address this hypothesis we carried out the following studies. 1. We characterized alterations of ZnT-1, ZnT-4 and ZnT-6 in the brain of preclinical AD (PCAD) subjects, who show no overt clinical manifestations of AD but demonstrate significant AD pathology at autopsy. 2. We identified the presence of ZnT-2 in human brain and compared protein levels in the brains of subjects with PCAD, mild cognitive impairment (MCI), early (EAD), and late-stage AD (LAD) to those in age matched normal control (NC) subjects. 3. We examined the relationship between protein levels of ZnT-1, ZnT-2, ZnT-4, ZnT-6 and Aβ produced by H4 human neuroglioma cells (H4-APP) transfected to overexpress amyloid precursor protein (APP), treated with short interfering RNA (siRNA) against each ZnT. Our data show a significant decrease (P < 0.05) of ZnT-1 and a significant increase of ZnT-6 in hippocampus/parahippo-campal gyrus (HPG) of PCAD subjects. In PCAD cerebellum (CER) the data show a significant increase of ZnT-4 and ZnT-6 compared to NC subjects. Levels of ZnT-2 were also significantly decreased in HPG of PCAD subjects compared to NC subjects. In addition, levels of ZnT-2 were significantly (P < 0.05) elevated in SMTG of PCAD and MCI subjects, compared to NC subjects. ZnT-2 was significantly (P < 0.05) elevated in HPG of EAD and LAD, and in SMTG of LAD brains, but was significantly (P < 0.05) decreased in LAD CER compared to NC subjects. siRNA mediated attenuation of each ZnT protein studied (ZnT-2, ZnT-4 and ZnT-6) led to significantly (P < 0.05) decreased production of Aβ compared to controls. Our results suggest alterations in Zn transport may play a role in Aβ processing and contribute to the neuropathology of AD.

Introdução: Perda de memória é um dos sintomas mais comuns em pacientes nos estágios iniciais da doença de Alzheimer; esses déficits são um reflexo do envolvimento da formação do hipocampo. O BDNF tem sido relacionado com a plasticidade do hipocampo. Neste sentido, as combinações de biomarcadores, como, por exemplo, a volumetria do hipocampo, pode apresentar um maior valor preditivo para diferenciar doença de Alzheimer do envelhecimento normal em pacientes com comprometimento cognitivo leve. Objetivo: A presente tese de doutorado teve como objetivo avaliar os níveis séricos do BDNF e o volume do hipocampo em pacientes com demência devido à doença de Alzheimer, Comprometimento Cognitivo Leve (CCL) e idosos saudáveis. Métodos: Para realização do estudo foram selecionados 10 idosos saudáveis, 10 CCL e 13 pacientes com demência devido à doença de Alzheimer pelos critérios NIA-AA. Todos participantes foram submetidos a uma avaliação cognitiva. Para as análises do BDNF, foi utilizado método de ELISA e para as análises de volumetria do hipocampo as imagens foram obtidas por meio de equipamento de ressonância de 1.5T e os volumes obtidos por meio do programa NeuroQuant®. Resultados: Idosos saudáveis apresentaram níveis séricos mais elevados de BDNF do que os CCL e pacientes com demência. O grupo de pacientes com demência apresentou menor volume total do hipocampo do que os idosos saudáveis e os CCL. Não houve correlação significativa do BDNF sérico com volume do hipocampo. Conclusão: Considerando nossos resultados em conjunto (baixos níveis de BDNF nos grupos CCL e demência devido à DA e menor volume do hipocampo na demência devido à AD), podemos supor que a diminuição dos níveis de BDNF ocorre antes da lesão neuronal expressa pela redução do hipocampo. / Introduction: Memory impairment is the most common symptom in patients in the early stages of Alzheimer's disease; this deficit is a reflection of the involvement of the hippocampal formation. BDNF has been linked to the hippocampal plasticity. Combinations of biomarkers, such as the hippocampal volumetry may have higher predictive value for differentiating Alzheimer's disease from normal aging in patients with mild cognitive impairment. Objective: The objective of present thesis was to evaluate serum levels of BDNF and hippocampal volume in patients with Mild Cognitive Impairment (MCI) and dementia due to Alzheimer's disease, and healthy elderly participants. Method: Ten healthy elderly subjects, 10 MCI and 13 patients with dementia due to Alzheimer's Disease (NIA-AA criteria) were selected for the study. All participants were assessed cognitively. The ELISA method was used for BDNF analysis, and the analysis of hippocampal volumetric images were acquired with 1.5T magnetic resonance equipment and volumes obtained with NeuroQuant® program. Results: Healthy elderly had higher BDNF serum levels than MCI and dementia due to AD patients. The group of dementia patients had lower total hippocampal volume than MCI and healthy elderly participants. No significant correlation between serum BDNF and hippocampal volume was observed. Conclusion: Taking our results together (lower BDNF levels in MCI and dementia due to AD and smaller hippocampal volume in dementia due to AD) we can hypothesize that the decrease of BDNF may start before the establishment of neuronal injury expressed by the hippocampal reduction.

A linhagem Wistar Audiogenic Rat (WAR), um modelo genético de epilepsia, apresenta diversas comorbidades. Dados prévios demonstraram níveis cerebrais elevados de proteína tau fosforilada (pTau), uma das principais características anatomopatológicas da doença de Alzheimer (DA). Estudos recentes sugerem que a DA seja um terceiro tipo de diabetes. O objetivo deste estudo foi validar a linhagem WAR como um possível modelo para o estudo da DA relacionada com alterações na regulação da via de sinalização da insulina. Para isso, foram selecionados 63 animais machos adultos e envelhecidos (n=28 Wistars, 14 Wistars Hannover e 21 WARs). Os animais foram submetidos ao labirinto aquático de Morris (LAM). Após o LAM, foram eutanasiados e tiveram seus hipocampos dissecados. Avaliou-se a expressão de betaamilóide (A?), tau, pTau (S396), receptor de insulina (IR), receptor de insulina -1 fosforilado em serina 312 (pIRS-1 S312) e glicogênio sintase quinase 3 alfa e beta (pGSK-3?/? S21/9) fosforiladas pelo método de Western blotting nos hipocampos ventral (HV) e dorsal (HD) e/ou confirmados por imunoistoquímica. Os animais da linhagem WAR adultos e envelhecidos apresentaram maior latência para encontrar a plataforma de fuga durante o período de treinamento e maioria dos retestes no LAM. Os animais WAR adultos apresentaram diminuição da expressão de A? e IR no HV, aumento da expressão de pGSK-3?/? (S21/9) no HD e aumento do número de células positivas para pIRS-1 (S312) no hipocampo. Os animais envelhecidos apresentaram aumento da expressão de pTau (S396) nos HV e HD, diminuição de A? e IR no HV e aumento da expressão de pGSK-3?/? (S21/9) nos HV e HD. Apesar de os animais WAR terem sido selecionados geneticamente para estudar epilepsia, nossos achados sugerem a coexistência de alterações comportamentais e moleculares características de DA e resistência central à insulina, podendo se somar ao fenótipo inicialmente selecionado. Portanto, outros estudos moleculares e histológicos são necessários para melhor compreensão dos dados encontrados e para possível validação da linhagem WAR como um modelo para estudar a relação entre DA e resistência central à insulina. / The Wistar Audiogenic Rat (WAR), a genetic model of epilepsy, presents many comorbidities. Previous data suggested elevated brain levels of phosphorylated tau (pTau), one of the the main hallmarks of Alzheimer\'s Disease (AD). Recent studies have hypothesized AD as the third type of diabetes. Therefore, the aim of this study was to validate the WAR strain as a model to study AD and correlate with alterations in the insulin signaling pathway regulation. To this end, we selected 63 male adult and aged animals (n=28 Wistars, 14 Wistars Hannover and 21 WARs). The animals were subjected to the Morris Water Maze (MWM) test. After the MWM, the rats were euthanized and their ventral (VH) and dorsal (DH) hippocampi were dissected for Western blot analysis in order to measure the expression levels of A?, tau, pTau (S396), insulin receptor (IR), alpha and beta phosphorylated glycogen synthase kinase 3 (pGSK-3?/? Ser21/9), and phospho-insulin receptor substrate-1 (pIRS-1 S312) in the ventral (VH) and dorsal hippocampus (DH) by means of the Western blotting method. The results were confirmed by immunohistochemical analysis. Both adult and aged WARs showed longer latency to find the escape platform during the learning period and most retests of the MWM. The adult WARs presented lower A? and IR expression in the VH, higher pGSK-3?/? (S21/9) expression in the DH, and higher levels of positive cells for pIRS-1 (S312) in the hippocampus. The aged WARs presented higher pTau (S396) expression in both VH and DH, lower A? and IR expression in the VH, higher pGSK-3?/? (S21/9) expression in both VH and DH. Although the WAR animals were primarily selected to study epilepsy, our findings in this study suggest the coexistence of an AD-like phenotype and central insulin resistance in addition to the initially selected epilepsy phenotype. Therefore, other molecular and histological studies are needed to better understand our data and to validate the WAR strain as a model to study the relationship between AD and central insulin resistance.

Introdução: Perda de memória é um dos sintomas mais comuns em pacientes nos estágios iniciais da doença de Alzheimer; esses déficits são um reflexo do envolvimento da formação do hipocampo. O BDNF tem sido relacionado com a plasticidade do hipocampo. Neste sentido, as combinações de biomarcadores, como, por exemplo, a volumetria do hipocampo, pode apresentar um maior valor preditivo para diferenciar doença de Alzheimer do envelhecimento normal em pacientes com comprometimento cognitivo leve. Objetivo: A presente tese de doutorado teve como objetivo avaliar os níveis séricos do BDNF e o volume do hipocampo em pacientes com demência devido à doença de Alzheimer, Comprometimento Cognitivo Leve (CCL) e idosos saudáveis. Métodos: Para realização do estudo foram selecionados 10 idosos saudáveis, 10 CCL e 13 pacientes com demência devido à doença de Alzheimer pelos critérios NIA-AA. Todos participantes foram submetidos a uma avaliação cognitiva. Para as análises do BDNF, foi utilizado método de ELISA e para as análises de volumetria do hipocampo as imagens foram obtidas por meio de equipamento de ressonância de 1.5T e os volumes obtidos por meio do programa NeuroQuant®. Resultados: Idosos saudáveis apresentaram níveis séricos mais elevados de BDNF do que os CCL e pacientes com demência. O grupo de pacientes com demência apresentou menor volume total do hipocampo do que os idosos saudáveis e os CCL. Não houve correlação significativa do BDNF sérico com volume do hipocampo. Conclusão: Considerando nossos resultados em conjunto (baixos níveis de BDNF nos grupos CCL e demência devido à DA e menor volume do hipocampo na demência devido à AD), podemos supor que a diminuição dos níveis de BDNF ocorre antes da lesão neuronal expressa pela redução do hipocampo. / Introduction: Memory impairment is the most common symptom in patients in the early stages of Alzheimer's disease; this deficit is a reflection of the involvement of the hippocampal formation. BDNF has been linked to the hippocampal plasticity. Combinations of biomarkers, such as the hippocampal volumetry may have higher predictive value for differentiating Alzheimer's disease from normal aging in patients with mild cognitive impairment. Objective: The objective of present thesis was to evaluate serum levels of BDNF and hippocampal volume in patients with Mild Cognitive Impairment (MCI) and dementia due to Alzheimer's disease, and healthy elderly participants. Method: Ten healthy elderly subjects, 10 MCI and 13 patients with dementia due to Alzheimer's Disease (NIA-AA criteria) were selected for the study. All participants were assessed cognitively. The ELISA method was used for BDNF analysis, and the analysis of hippocampal volumetric images were acquired with 1.5T magnetic resonance equipment and volumes obtained with NeuroQuant® program. Results: Healthy elderly had higher BDNF serum levels than MCI and dementia due to AD patients. The group of dementia patients had lower total hippocampal volume than MCI and healthy elderly participants. No significant correlation between serum BDNF and hippocampal volume was observed. Conclusion: Taking our results together (lower BDNF levels in MCI and dementia due to AD and smaller hippocampal volume in dementia due to AD) we can hypothesize that the decrease of BDNF may start before the establishment of neuronal injury expressed by the hippocampal reduction.

The dementias are persistent or progressive disorders affecting more than one cognitive domain that interfere with an individual’s ability to function at work or home, and represent a decline from a previous level of function. In this thesis I consider the neurophysiology of dementia at a number of levels. I investigate the ways in which the connectivity and function of the brain predisposes to the specific focal patterns of neurodegeneration seen in the various dementias. I aim to identify the mesoscopic changes that occur in individuals with neurodegeneration and how these relate to their cognitive difficulties. I show how, by assessing patients in whom there is focal disruption of brain networks and observing the outcomes in comparison to controls, I can gain insight into the mechanisms by which the normal brain makes predictions and processes language. In Chapter 1, I set the scene for the focussed experimental investigations of model diseases by beginning with an introductory, clinically-focussed review that sets out the features, aetiology, management, epidemiology and prognosis of the dementias. This places these model diseases in the context of the broader clinical challenge posed by the dementias. In Chapter 2, I turn to ‘prototypical’ model diseases that represent neurodegenerative tauopathies with predominantly cortical (Alzheimer’s disease, AD) and subcortical (Progressive Supranuclear Palsy, PSP) disease burdens. I investigate the neurophysiological causes and consequences of Tau accumulation by combining graph theoretical analyses of resting state functional MR imaging and in vivo ‘Tau’ PET imaging using the ligand AV-1451. By relating Tau distribution to the functional connectome I provide in vivo evidence consistent with ‘prion-like’ trans-neuronal spread of Tau in AD but not PSP. This provides important validation of disease modification strategies that aim to halt or slow down the progression of AD by sequestration of pathological Tau in the synapse. In contrast, I demonstrate associations consistent with regional vulnerability to Tau accumulation due to metabolic demand and a lack of trophic support in PSP but not AD. With a cross-sectional approach, using Tau burden as a surrogate marker of disease severity, I then go on to show how the changes in functional connectivity that occur as disease progresses account for the contrasting cognitive phenotypes in AD and PSP. In advancing AD, functional connectivity across the whole brain becomes increasingly random and disorganised, accounting for symptomatology across multiple cognitive domains. In advancing PSP, by contrast, disrupted cortico-subcortical and cortico-brainstem interactions meant that information transfer passed through a larger number of cortical nodes, reducing closeness centrality and eigenvector centrality, while increasing weighted degree, clustering, betweenness centrality and local efficiency. Together, this resulted in increasingly modular processing with inter-network communication taking less direct paths, accounting for the bradyphrenia characteristic of the ‘subcortical dementias’. From chapter 3 onwards, I turn to the in-depth study of a model disease called non-fluent variant Primary Progressive Aphasia (nfvPPA). This disease has a clear clinical phenotype of speech apraxia and agrammatism, associated with a focal pattern of mild atrophy in frontal lobes. Importantly, general cognition is usually well preserved until late disease. In chapter 3 itself, I relate an experiment in which patients with nfvPPA and matched controls performed a receptive language task while having their brain activity recorded with magnetoencephalography. I manipulated expectations and sensory detail to explore the role of top-down frontal contributions to predictive processes in speech perception. I demonstrate that frontal neurodegeneration led to inflexible and excessively precise predictions, and that fronto-temporal interactions play a causal role in reconciling prior predictions with degraded sensory signals. The discussion here concentrates on the insights provided by neurodegenerative disease into the normal function of the brain in processing language. Overall, I demonstrate that higher level frontal mechanisms for cognitive and behavioural flexibility make a critical functional contribution to the hierarchical generative models underlying speech perception In chapter 4, I precisely define the sequence processing and statistical learning abilities of patients with nfvPPA in comparison to patients with non-fluent aphasia due to stroke and neurological controls. I do this by exposing participants to a novel, mixed-complexity artificial grammar designed to assess processing of increasingly complex sequencing relationships, and then assessing the degree of implicit rule learning. I demonstrate that agrammatic aphasics of two different aetiologies are not disproportionately impaired on complex sequencing relationships, and that the learning of phonological and non-linguistic sequences occurs independently in health and disease. In chapter 5, I summarise the synergies between the experimental chapters, and explain how I have applied a systems identification framework to a diverse set of experimental methods, with the common goal of defining the physiology of dementia. I then return to the results of chapter 3 with a clinical focus to explain how inflexible predictions can account for subjective speech comprehension difficulties, auditory processing abnormalities and (in synthesis with chapter 4) receptive agrammatism in nfvPPA. Overall, this body of work has contributed to knowledge in several ways. It has achieved its tripartite aims by: 1) Providing in vivo evidence consistent with theoretical models of trans-neuronal Tau spread (chapter 2), and a comprehensive clinical account of the previously poorly-understood receptive symptomatology of nfvPPA (chapter 5), thus demonstrating that systems neuroscience can provide a translational bridge between the molecular biology of dementia and clinical trials of therapies and medications. In this way, I begin to disentangle the network-level causes of neurodegeneration from its consequences. 2) Providing evidence for a causal role for fronto-temporal interactions in language processing (chapter 3), and demonstrating domain separation of statistical learning between linguistic and non-linguistic sequences (chapter 4), thus demonstrating that studies of patients with neurodegenerative disease can further our understanding of normative brain function. 3) Successfully integrating neuropsychology, behavioural psychophysics, functional MRI, structural MRI, magnetoencephalography and computational modelling to provide comprehensive research training, as the platform for a future research programme in the physiology of dementia.

Introdução: Perda de memória é um dos sintomas mais comuns em pacientes nos estágios iniciais da doença de Alzheimer; esses déficits são um reflexo do envolvimento da formação do hipocampo. O BDNF tem sido relacionado com a plasticidade do hipocampo. Neste sentido, as combinações de biomarcadores, como, por exemplo, a volumetria do hipocampo, pode apresentar um maior valor preditivo para diferenciar doença de Alzheimer do envelhecimento normal em pacientes com comprometimento cognitivo leve. Objetivo: A presente tese de doutorado teve como objetivo avaliar os níveis séricos do BDNF e o volume do hipocampo em pacientes com demência devido à doença de Alzheimer, Comprometimento Cognitivo Leve (CCL) e idosos saudáveis. Métodos: Para realização do estudo foram selecionados 10 idosos saudáveis, 10 CCL e 13 pacientes com demência devido à doença de Alzheimer pelos critérios NIA-AA. Todos participantes foram submetidos a uma avaliação cognitiva. Para as análises do BDNF, foi utilizado método de ELISA e para as análises de volumetria do hipocampo as imagens foram obtidas por meio de equipamento de ressonância de 1.5T e os volumes obtidos por meio do programa NeuroQuant®. Resultados: Idosos saudáveis apresentaram níveis séricos mais elevados de BDNF do que os CCL e pacientes com demência. O grupo de pacientes com demência apresentou menor volume total do hipocampo do que os idosos saudáveis e os CCL. Não houve correlação significativa do BDNF sérico com volume do hipocampo. Conclusão: Considerando nossos resultados em conjunto (baixos níveis de BDNF nos grupos CCL e demência devido à DA e menor volume do hipocampo na demência devido à AD), podemos supor que a diminuição dos níveis de BDNF ocorre antes da lesão neuronal expressa pela redução do hipocampo. / Introduction: Memory impairment is the most common symptom in patients in the early stages of Alzheimer's disease; this deficit is a reflection of the involvement of the hippocampal formation. BDNF has been linked to the hippocampal plasticity. Combinations of biomarkers, such as the hippocampal volumetry may have higher predictive value for differentiating Alzheimer's disease from normal aging in patients with mild cognitive impairment. Objective: The objective of present thesis was to evaluate serum levels of BDNF and hippocampal volume in patients with Mild Cognitive Impairment (MCI) and dementia due to Alzheimer's disease, and healthy elderly participants. Method: Ten healthy elderly subjects, 10 MCI and 13 patients with dementia due to Alzheimer's Disease (NIA-AA criteria) were selected for the study. All participants were assessed cognitively. The ELISA method was used for BDNF analysis, and the analysis of hippocampal volumetric images were acquired with 1.5T magnetic resonance equipment and volumes obtained with NeuroQuant® program. Results: Healthy elderly had higher BDNF serum levels than MCI and dementia due to AD patients. The group of dementia patients had lower total hippocampal volume than MCI and healthy elderly participants. No significant correlation between serum BDNF and hippocampal volume was observed. Conclusion: Taking our results together (lower BDNF levels in MCI and dementia due to AD and smaller hippocampal volume in dementia due to AD) we can hypothesize that the decrease of BDNF may start before the establishment of neuronal injury expressed by the hippocampal reduction.

No description available.

A Doença de Alzheimer (DA) é a principal causa de demência na população idosa e tende a se tornar um grave problema de saúde pública com o aumento da expectativa de vida da população mundial. A perda progressiva de memória, principal sintoma da demência em pacientes com DA, é atribuída a danos sinápticos e à perda neuronal desencadeadas pelo desequilíbrio entre a produção e a depuração do peptídeo A?. Evidências surgidas nos últimos 20 anos apontam os oligômeros solúveis de A? (A?O), produtos de agregação do peptídeo A?, como as principais espécies neurotóxicas na DA. Por conta disso, e também pela ausência de métodos diagnósticos pre-mortem e tratamento eficientes para essa demência, a busca por anticorpos conformacionais específicos para A?O está em ascensão. Testes clínicos com IgG anti-A? resultaram em efeitos colaterais inflamatórios mediados pela porção não variável Fc. Então, anticorpos conformacionais artificiais do tipo scFv, desprovidos de porção Fc, foram selecionados contra A?O. Dentre eles, está NUsc1, que é neuroprotetor contra A?O em cultura primária de neurônios. Neste trabalho, avaliamos a toxicidade de A?Os na linhagem de neuroblastoma humano SH-SY5Y diferenciada em neurônios maduros e comparamos a neuroproteção conferida por diferentes anticorpos contra A?Os, por ensaio de viabilidade celular com MTT. Também avaliamos a especificidade de NUsc1 por A?O comparativamente a lisozima monomérica e oligomérica em ensaio de ELISA, já que outros anticorpos conformacionais reconhecem epítopo compartilhado por estados oligoméricos de outras proteínas amiloidogênicas. Para a validação de células SH-SY5Y como modelo in vitro de neurônios maduros, a diferenciação foi induzida com ácido retinoico e BDNF e as células foram marcadas para as proteínas MAP2 e NeuN em ensaio de imunofluorescência. Células submetidas ao protocolo de diferenciação apresentaram aumento dos níveis dessas proteínas, mudança morfológica condizente com o esperado na maturação neuronal. Posteriormente, o desafio da cultura com A?O indicou morte celular dose-dependente e reversão desta morte segundo a dose administrada dos anticorpos 6E10 e NU-4. Obtivemos um sinal cerca de 400 vezes maior no reconhecimento de A?O por NUsc1 que para oligômeros de lisozima, quando presentes na mesma concentração, indicando forte especificidade de NUsc1 por A?O. Além disso, NUsc1 purificado em sistema de gelfiltração em HPLC não apresenta citotoxicidade em concentração equivalente a dos anticorpos 6E10 e NU-4 em ensaios de neuroproteção em cultura de SH-SY5Y diferenciada, sugerindo que, se NUsc1 for tão eficiente quanto estas IgG\'s, este poderá ser usado em dose não citotóxica. Portanto, podemos concluir que NUsc1 apresenta grande potencial como ferramenta diagnóstica e terapêutica para a DA, mas que mais experimentos para expandir sua validação e potencial ainda são necessários. / Alzheimer\'s Disease (AD) is the leading cause of dementia in the elderly population and tends to become a serious public health problem with increasing life expectancy of the world\'s population. Progressive memory loss, the main symptom of dementia in patients with AD, is attributed to synaptic damage and neuronal loss triggered by imbalance between production and clearance of the A? peptide. Evidence from the last 20 years indicates that soluble A? oligomers (A?O), A? peptide aggregation products, as the main neurotoxic species in AD. Because of this, and also because of the absence of efficient pre-mortem diagnostic and treatment methods for this dementia, the search for conformational antibodies specific for A?O is on the rise. Clinical tests with anti-A? IgG\'s resulted in inflammatory side effects mediated by the non-variable Fc portion. Then, artificial conformational antibodies of the scFv type, lacking the Fc portion, were selected against A?O. Among them is NUsc1, which is neuroprotective against A?O in primary neuronal culture. In this work, we evaluated the toxicity of A?Os in the differentiated SH-SY5Y human neuroblastoma line in mature neurons and compared the neuroprotection conferred by different antibodies against A?O types by MTT cell viability assay. We also evaluated the specificity of NUsc1 for A?O compared to monomeric and oligomeric lysozyme in the ELISA assay, since other conformational antibodies recognize epitope shared by oligomeric states of other amyloidogenic proteins. For the validation of SH-SY5Y cells as an in vitro model of mature neurons, differentiation was induced with retinoic acid and BDNF and the cells were labeled for MAP2 and NeuN proteins in immunofluorescence assay. Cells submitted to the differentiation protocol presented increased levels of these proteins, a morphological change consistent with the expected neuronal maturation. Subsequently, the challenge of culture with A?O indicated dose-dependent cell death and reversion of this death according to the administered dose of 6E10 and NU-4 antibodies. We obtained a 400-fold higher signal in the recognition of A?O by NUsc1 than for lysozyme oligomers, when present at the same concentration, indicating strong specificity of A?O by NUsc1. In addition, NUsc1 purified on HPLC gel-filtration system does not exhibit cytotoxicity at concentration equivalent to 6E10 and NU-4 antibodies in neuroprotection assays in differentiated SH-SY5Y culture, suggesting that, if NUsc1 is as efficient as these IgG\'s, it may be used in a non-cytotoxic dose. Therefore, we can conclude that NUsc1 presents great potential as a diagnostic and therapeutic tool for AD, but that further experiments to expand its validation and potential are still necessary.

A Doença de Alzheimer (DA) é a principal causa de demência na população idosa e tende a se tornar um grave problema de saúde pública com o aumento da expectativa de vida da população mundial. A perda progressiva de memória, principal sintoma da demência em pacientes com DA, é atribuída a danos sinápticos e à perda neuronal desencadeadas pelo desequilíbrio entre a produção e a depuração do peptídeo A?. Evidências surgidas nos últimos 20 anos apontam os oligômeros solúveis de A? (A?O), produtos de agregação do peptídeo A?, como as principais espécies neurotóxicas na DA. Por conta disso, e também pela ausência de métodos diagnósticos pre-mortem e tratamento eficientes para essa demência, a busca por anticorpos conformacionais específicos para A?O está em ascensão. Testes clínicos com IgG anti-A? resultaram em efeitos colaterais inflamatórios mediados pela porção não variável Fc. Então, anticorpos conformacionais artificiais do tipo scFv, desprovidos de porção Fc, foram selecionados contra A?O. Dentre eles, está NUsc1, que é neuroprotetor contra A?O em cultura primária de neurônios. Neste trabalho, avaliamos a toxicidade de A?Os na linhagem de neuroblastoma humano SH-SY5Y diferenciada em neurônios maduros e comparamos a neuroproteção conferida por diferentes anticorpos contra A?Os, por ensaio de viabilidade celular com MTT. Também avaliamos a especificidade de NUsc1 por A?O comparativamente a lisozima monomérica e oligomérica em ensaio de ELISA, já que outros anticorpos conformacionais reconhecem epítopo compartilhado por estados oligoméricos de outras proteínas amiloidogênicas. Para a validação de células SH-SY5Y como modelo in vitro de neurônios maduros, a diferenciação foi induzida com ácido retinoico e BDNF e as células foram marcadas para as proteínas MAP2 e NeuN em ensaio de imunofluorescência. Células submetidas ao protocolo de diferenciação apresentaram aumento dos níveis dessas proteínas, mudança morfológica condizente com o esperado na maturação neuronal. Posteriormente, o desafio da cultura com A?O indicou morte celular dose-dependente e reversão desta morte segundo a dose administrada dos anticorpos 6E10 e NU-4. Obtivemos um sinal cerca de 400 vezes maior no reconhecimento de A?O por NUsc1 que para oligômeros de lisozima, quando presentes na mesma concentração, indicando forte especificidade de NUsc1 por A?O. Além disso, NUsc1 purificado em sistema de gelfiltração em HPLC não apresenta citotoxicidade em concentração equivalente a dos anticorpos 6E10 e NU-4 em ensaios de neuroproteção em cultura de SH-SY5Y diferenciada, sugerindo que, se NUsc1 for tão eficiente quanto estas IgG\'s, este poderá ser usado em dose não citotóxica. Portanto, podemos concluir que NUsc1 apresenta grande potencial como ferramenta diagnóstica e terapêutica para a DA, mas que mais experimentos para expandir sua validação e potencial ainda são necessários. / Alzheimer\'s Disease (AD) is the leading cause of dementia in the elderly population and tends to become a serious public health problem with increasing life expectancy of the world\'s population. Progressive memory loss, the main symptom of dementia in patients with AD, is attributed to synaptic damage and neuronal loss triggered by imbalance between production and clearance of the A? peptide. Evidence from the last 20 years indicates that soluble A? oligomers (A?O), A? peptide aggregation products, as the main neurotoxic species in AD. Because of this, and also because of the absence of efficient pre-mortem diagnostic and treatment methods for this dementia, the search for conformational antibodies specific for A?O is on the rise. Clinical tests with anti-A? IgG\'s resulted in inflammatory side effects mediated by the non-variable Fc portion. Then, artificial conformational antibodies of the scFv type, lacking the Fc portion, were selected against A?O. Among them is NUsc1, which is neuroprotective against A?O in primary neuronal culture. In this work, we evaluated the toxicity of A?Os in the differentiated SH-SY5Y human neuroblastoma line in mature neurons and compared the neuroprotection conferred by different antibodies against A?O types by MTT cell viability assay. We also evaluated the specificity of NUsc1 for A?O compared to monomeric and oligomeric lysozyme in the ELISA assay, since other conformational antibodies recognize epitope shared by oligomeric states of other amyloidogenic proteins. For the validation of SH-SY5Y cells as an in vitro model of mature neurons, differentiation was induced with retinoic acid and BDNF and the cells were labeled for MAP2 and NeuN proteins in immunofluorescence assay. Cells submitted to the differentiation protocol presented increased levels of these proteins, a morphological change consistent with the expected neuronal maturation. Subsequently, the challenge of culture with A?O indicated dose-dependent cell death and reversion of this death according to the administered dose of 6E10 and NU-4 antibodies. We obtained a 400-fold higher signal in the recognition of A?O by NUsc1 than for lysozyme oligomers, when present at the same concentration, indicating strong specificity of A?O by NUsc1. In addition, NUsc1 purified on HPLC gel-filtration system does not exhibit cytotoxicity at concentration equivalent to 6E10 and NU-4 antibodies in neuroprotection assays in differentiated SH-SY5Y culture, suggesting that, if NUsc1 is as efficient as these IgG\'s, it may be used in a non-cytotoxic dose. Therefore, we can conclude that NUsc1 presents great potential as a diagnostic and therapeutic tool for AD, but that further experiments to expand its validation and potential are still necessary.