O aumento da expectativa de vida da população mundial tem se associado com uma maior prevalência de doenças neurodegenerativas. A Doença de Alzheimer (DA) é a doença neurodegenerativa mais comum e a principal causa de demência em indivíduos com mais de 60 anos, sendo caracterizada por um declínio progressivo na memória e função mental dos pacientes. Esses sintomas são acompanhados por alterações histopatológicas no cérebro desses indivíduos, incluindo a presença de uma grande quantidade de placas senis, formadas pela deposição do peptídeo beta-amiloide (Aβ), e de emaranhados neurofibrilares formados pela hiperfosforilação da proteína Tau. Estudos indicam que a deposição do Aβ é uma das principais responsáveis pelo desenvolvimento da DA, causando dano neuronal através da ativação de várias vias pró-apoptóticas e dando origem aos sintomas de demência típicos dessa doença. Até o momento, não existem tratamentos eficazes para o combate à DA, de forma que a maior parte das intervenções farmacológicas é destinada apenas ao tratamento de alguns de seus sintomas. A proteína translocadora (TSPO) se localiza em pontos de contato entre as membranas mitocondriais interna e externa e está relacionada com o transporte de colesterol para o interior da mitocôndria e com a regulação da esteroidogênese e da apoptose. Estudos mostram que ligantes da TSPO apresentam efeitos neuroprotetores em diferentes modelos experimentais de lesão cerebral e doenças neurodegenerativas. Especificamente em relação à DA, um estudo indicou que o 4’-clorodiazepam (4’-CD), um ligante da TSPO, apresenta efeitos neuroprotetores em um modelo animal dessa doença, sendo um possível candidato para o seu tratamento. Dessa forma, o objetivo desse estudo foi verificar o efeito neuroprotetor do 4’-CD em diferentes modelos in vitro de toxicidade induzida pelo Aβ, além de seus efeitos sobre o desenvolvimento de neurônios hipocampais. Inicialmente, demonstramos que o 4’-CD reduziu a morte celular de células SH-SY5Y expostas a um modelo de toxicidade induzida pela administração de Aβ. Esses efeitos estiveram associados com a redução da expressão da proteína pró-apoptótica Bax e com um aumento da expressão da survivina, uma proteína anti-apoptótica. A expressão das proteínas Bcl-xl e procaspase-3, por outro lado, não foi alterada pelos tratamentos. Posteriormente, estudamos os efeitos neuroprotetores do 4’-CD contra a toxicidade induzida pela administração do Aβ em culturas organotípicas de hipocampo. Nesses experimentos, foi demonstrado que o 4’-CD reduz a morte celular de culturas organotípicas de hipocampo expostas ao Aβ através de um aumento na expressão da enzima SOD, sem alterar, no entanto, a expressão das proteínas Akt e procaspase-3. Por fim, foi avaliado o efeito do 4’-CD sobre o desenvolvimento de culturas primárias de neurônios hipocampais de camundongos machos e fêmeas. Foi observado que as culturas de neurônios hipocampais das fêmeas apresentaram um desenvolvimento mais rápido do que as dos machos. O 4’-CD acelerou a maturação e aumentou a ramificação neurítica dos neurônios hipocampais dos machos, mas não exerceu qualquer efeito sobre os neurônios das fêmeas. Em suma, foi observado que o 4’-CD apresenta efeitos neuroprotetores contra o Aβ em células SH-SY5Y e em culturas organotípicas do hipocampo, apresentando-se como um fármaco em potencial para o tratamento da DA. Além disso, foi observado que o 4’-CD exerceu um efeito dependente do sexo sobre o desenvolvimento de culturas primárias de neurônios hipocampais, estimulando o desenvolvimento e a ramificação neurítica de neurônios hipocampais de machos, mas não de fêmeas. / The increase in life expectancy of the world population has been associated with a higher prevalence of neurodegenerative diseases. The Alzheimer’s Disease (AD) is the most common neurodegenerative disorder and the main cause of dementia among people over 60 years, being characterized by a progressive decline in the memory and mental function of the patients. These symptoms are associated with histopathological changes in the brain of these patients, including the presence of senile plaques, formed by the deposition of amyloid-beta (Aβ), and neurofibrillary tangles, which are related to the hyperphosphorylation of Tau protein. Studies indicate that Aβ deposition is a major contributor to AD progression, promoting neuronal damage through the activation of different pro-apoptotic pathways and giving rise to the typical dementia symptoms of this disease. To date, there are no effective treatments for AD, so that most of the pharmacological intervention is intended for the treatment of some of its symptoms. The translocator protein (TSPO) is located in contact sites between the outer and the inner mitochondrial membranes and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and apoptosis. Studies show that TSPO ligands present neuroprotective effects in different experimental models of brain injury and neurodegenerative diseases. Specifically regarding AD, a study indicated that 4’-chlorodiazepam (4’-CD), a TSPO ligand, is neuroprotective in an animal model of this disease, being a possible candidate for its treatment. Therefore, the aim of this study was to evaluate the neuroprotective effect of 4’-CD in different experimental models of Aβ- induced neurotoxicity in vitro, as well as its effects on the development of hipocampal neurons. First, it was demonstrated that 4’-CD decreased the cell death of SH-SY5Y cells exposed to the Aβ. This effect was associated with the inhibition of the Aβ-induced upregulation of Bax, a pro-apoptotic protein, and downregulation of survivin, a prosurvival protein. On the other hand, the expression of Bcl-xl and procaspase-3 was not change by the treatments. After, it was studied the neuroprotective effects of 4’-CD against Aβ in organotypic hipocampal cultures. In these experiments, it was shown that 4’-CD decreases the cell death of organotypic hippocampal slices exposed to the Aβ by increasing the protein expression of SOD, but without changing the expression of Akt and procaspase-3. Finally, due to the importance of the processes of neuronal development and maturation in the regeneration of CNS after injury, it was evaluated the effect of 4’-CD on the development of primary hippocampal neurons of male and female mice. It was observed that female primary hippocampal neurons presented an increased rate of development than male neurons. 4’-CD stimulated the development and increased the neuritic branching of male but not from female neurons. In summary, it was observed that 4’-CD presented a neuroprotective effect against Aβ in SH-SY5Y cells and in rat organotypical hippocampal slices, presenting itself as a promising agent for the treatment of AD. Also, it was observed that 4’-CD modulates the development of hippocampal neurons in a sex-dependent manner, stimulating the development of male but not from female cells.

Cheng, Wai Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 76-88). / Abstract also in Chinese. / Acknowledgement --- p.i / 摘要 --- p.iii / List of Abbreviations --- p.iv / List of Figures --- p.vi / List of Tables --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- FE65 --- p.1 / Chapter 1.1.1 --- FE65 Protein Family and Their Structures --- p.2 / Chapter 1.1.1.2 --- PTB domains --- p.5 / Chapter 1.1.2 --- Expression Pattern of FE65 Proteins --- p.6 / Chapter 1.1.3 --- FE65 Family-Transgenic Animals --- p.7 / Chapter 1.1.4 --- Interacting Partners of FE65 --- p.8 / Chapter 1.1.4.1 --- "APP, APLPl and APLP2" --- p.9 / Chapter 1.1.4.2 --- LRP1 and ApoEr2 --- p.10 / Chapter 1.1.4.3 --- c-Abl --- p.11 / Chapter 1.1.4.4 --- Mena and EVL --- p.11 / Chapter 1.1.4.5 --- Tip60 --- p.12 / Chapter 1.1.4.6 --- SET --- p.12 / Chapter 1.1.4.7 --- Estrogen Receptor a --- p.13 / Chapter 1.1.4.8 --- Teashirt --- p.13 / Chapter 1.1.4.9 --- CP2/LSF/LBP1 --- p.13 / Chapter 1.1.4.10 --- Dexra sl --- p.14 / Chapter 1.1.4.11 --- P2X2-receptor subunit --- p.14 / Chapter 1.1.4.12 --- Tau --- p.15 / Chapter 1.1.4.13 --- Notchl --- p.15 / Chapter 1.1.4.14 --- Alcadein --- p.16 / Chapter 1.1.4.15 --- CD95/Fas/Apo -1 ligand --- p.16 / Chapter 1.1.4.16 --- p68 subunit of pre -mRNA cleavage and polyadenylation factor Im (p68 CFIm) --- p.17 / Chapter 1.1.4.17 --- Ataxinl --- p.17 / Chapter 1.1.5.1 --- FE65 as an adaptor protein --- p.20 / Chapter 1.1.5.2 --- FE65 and Alzheimer´ةs disease --- p.20 / Chapter 1.1.5.3 --- Transcriptional / Post-transcriptional regulation --- p.22 / Chapter 1.1.5.4 --- Apoptosis and cell cycle regulation --- p.23 / Chapter 1.1.5.5 --- Neuronal positioning and cell migration --- p.23 / Chapter 1.1.5.6 --- Learning and memory --- p.25 / Chapter 1.2 --- Objectives --- p.26 / Chapter Chapter 2 --- Investigation of the interaction between FE65 and Arf6 --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- DNA contructs --- p.27 / Chapter 2.1.2 --- Cell culture --- p.27 / Chapter 2.1.3 --- Immunoblotting --- p.28 / Chapter 2.1.4 --- Miscellaneous --- p.28 / Chapter 2.2 --- Methods --- p.29 / Chapter 2.2.1 --- Preparation of Escherichia coli competent cells --- p.29 / Chapter 2.2.2 --- DNA preparation with Intron Plasmid DNA --- p.30 / Chapter 2.2.3 --- DNA preparation with Macherey-Nagel NucleoBond Xtra Midi --- p.30 / Chapter 2.2.4 --- DNA preparation by the alkaline lysis method --- p.31 / Chapter 2.2.5 --- Spectrophotometric analysis of DNA --- p.32 / Chapter 2.2.6 --- Agarose gel electrophoresis --- p.32 / Chapter 2.2.7 --- Cell culture and transfection --- p.33 / Chapter 2.2.8 --- Bacterial GST-pull down assay --- p.33 / Chapter 2.2.9 --- GST-pull down assay for testing direct interaction between FE65 and Arf6 --- p.34 / Chapter 2.2.10 --- Mammalian GST-pull down assay --- p.35 / Chapter 2.2.11 --- Immunoprecipitation --- p.36 / Chapter 2.2.12 --- SDS-PAGE --- p.36 / Chapter 2.2.13 --- Immunoblotting --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Interaction between Arf6 and FE65 --- p.40 / Chapter 2.3.2 --- Determination of the interacting domain of FE65 with Arf6 --- p.43 / Chapter 2.3.3 --- Determination if FE65 and Arf6 interact directly --- p.45 / Chapter Chapter 3 --- Production of Antisera against Arf6 and Immunostaining of FE65-Arf6 --- p.47 / Chapter 3.1 --- Materials --- p.47 / Chapter 3.1.1 --- Protein expression and purification --- p.47 / Chapter 3.1.2 --- Immunization and harvest of antisera --- p.48 / Chapter 3.1.3 --- Immunostaining --- p.48 / Chapter 3.2 --- Methods --- p.48 / Chapter 3.2.1 --- Protein expression and purification --- p.48 / Chapter 3.2.2 --- Bradford assay --- p.50 / Chapter 3.2.3 --- Immunization --- p.50 / Chapter 3.2.4 --- Antibody purification --- p.51 / Chapter 3.2.5 --- Immunostaining --- p.52 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- Recombinant Arf6 expression and purification --- p.53 / Chapter 3.3.2 --- Titering of antisera --- p.57 / Chapter 3.3.3 --- Determination of antisera specificity --- p.59 / Chapter Chapter 4 --- Discussion --- p.68 / Chapter Chapter 5 --- Future Perspectives --- p.73 / References --- p.76

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

No description available.

<p>Heparan sulfate (HS) is a linear polysaccharide, located on the surface and in the extracellular matrix of most cells, that regulates functions of numerous proteins. HS-protein interaction is mainly mediated by sulfate groups found in N-sulfated (NS) regions of the HS, but may also involve rare HS substituents such as N-unsubstituted glucosamine (GlcNH₂) residues. The location of GlcNH₂ in an HS-epitope recognized by the monoclonal antibody 10E4, that specifically stains the prion lesions in scrapie-infected murine brain, suggests an involvement of GlcNH₂ in prion disease and other amyloid-related disorders. HS in general is strongly associated with amyloidosis, including Alzheimer’s disease (AD). Therefore, the aims of this thesis were to structurally characterize GlcNH₂-containing HS sequences found in native tissues, to further study HS epitopes recognized by 10E4, and to investigate the possible role(s) of GlcNH₂ and other HS structures in binding to amyloid β peptide (Aβ) (core material in AD plaque lesions, also stained by 10E4).</p><p>The GlcNH₂ content (0.7-4% of total disaccharide units) varied between HS from different tissues. Most GlcNH₂ units were found in poorly modified N-acetylated (NA-) or NA/NS-domains, located toward the polysaccharide-protein linkage region.</p><p>Binding of human cerebral cortex HS to Aβ(1–40) monomers requires N-, 2- and 6-O-sulfation of HS, while binding to Aβ fibrils requires N- and 2-O-sulfation only. GlcNH₂ units do not appreciably contribute to interaction with Aβ. Aβ fibril-binding HS domains also bind to fibroblast growth factor 2 (FGF-2), indicating that Aβ (neurotoxic) and FGF-2 (neuroprotective) may compete for common binding sites in HS. However, Aβ had no effect on FGF-2-induced MAPK signaling in NIH 3T3 fibroblasts.</p><p>Continued studies on 10E4-antigenic HS epitope(s) showed that binding of 10E4 to the previously identified antigenic tetrasaccharide, ∆UA-GlcNH₂-GlcA-GlcNAc, requires the nonreducing hexuronic acid (∆UA) to be 4,5 unsaturated (induced by lyase cleavage), and thus is artificial. Further studies are needed to clarify the potential involvement of GlcNH₂ in 10E4-recognition of the native HS epitope(s).</p>

Heparan sulfate (HS) is a linear polysaccharide, located on the surface and in the extracellular matrix of most cells, that regulates functions of numerous proteins. HS-protein interaction is mainly mediated by sulfate groups found in N-sulfated (NS) regions of the HS, but may also involve rare HS substituents such as N-unsubstituted glucosamine (GlcNH2) residues. The location of GlcNH2 in an HS-epitope recognized by the monoclonal antibody 10E4, that specifically stains the prion lesions in scrapie-infected murine brain, suggests an involvement of GlcNH2 in prion disease and other amyloid-related disorders. HS in general is strongly associated with amyloidosis, including Alzheimer’s disease (AD). Therefore, the aims of this thesis were to structurally characterize GlcNH2-containing HS sequences found in native tissues, to further study HS epitopes recognized by 10E4, and to investigate the possible role(s) of GlcNH2 and other HS structures in binding to amyloid β peptide (Aβ) (core material in AD plaque lesions, also stained by 10E4). The GlcNH2 content (0.7-4% of total disaccharide units) varied between HS from different tissues. Most GlcNH2 units were found in poorly modified N-acetylated (NA-) or NA/NS-domains, located toward the polysaccharide-protein linkage region. Binding of human cerebral cortex HS to Aβ(1–40) monomers requires N-, 2- and 6-O-sulfation of HS, while binding to Aβ fibrils requires N- and 2-O-sulfation only. GlcNH2 units do not appreciably contribute to interaction with Aβ. Aβ fibril-binding HS domains also bind to fibroblast growth factor 2 (FGF-2), indicating that Aβ (neurotoxic) and FGF-2 (neuroprotective) may compete for common binding sites in HS. However, Aβ had no effect on FGF-2-induced MAPK signaling in NIH 3T3 fibroblasts. Continued studies on 10E4-antigenic HS epitope(s) showed that binding of 10E4 to the previously identified antigenic tetrasaccharide, ∆UA-GlcNH2-GlcA-GlcNAc, requires the nonreducing hexuronic acid (∆UA) to be 4,5 unsaturated (induced by lyase cleavage), and thus is artificial. Further studies are needed to clarify the potential involvement of GlcNH2 in 10E4-recognition of the native HS epitope(s).

Interactions between peptides and biological lipid membranes play a crucial role in many cellular processes such as in the mechanism behind Alzheimer’s disease where amyloid-beta peptide (Abeta)is thought to be a key component. The initial step of binding between a surface active peptide and its target membrane or membrane receptor can involve a non specific electrostatic association where positively charged amino acid residues and a negatively charged membrane surface interact. Here, the use of high resolution MAS NMR provides a highly sensitive and non perturbing way of studying the electrostatic potential present at lipid membrane surfaces and the changes resulting from the association of peptides. The interaction between pharmacologically relevant peptides and lipid membranes can also involve incorporation of the peptide into the membrane core and by complementing the NMR approach with differential scanning calorimetry (DSC) the hydrophobic incorporation can be studied in a non invasive way. By using 14N MAS NMR on biological lipid systems for the first time, in addition to 31P, 2H NMR and differential scanning calorimetry (DSC), gives a full picture of the changes all along the phospholipid following interactions at the membrane interface region. Being able to monitor the full length of the phospholipid enables us to differentiate between interactions related to either membrane surface association or hydrophobic core incorporation. This approach was used to establish that the interaction between nociceptin and negatively charged lipid membranes is electrostatic and hence that nociceptin can initially associate with a membrane surface before binding to its receptor. Also, it was found that Abeta can interact with phospholipid membranes via two types of interactions with fundamentally adverse effects. The results reveal that Abeta can associate with the surface of a neuronal membrane promoting accelerated aggregation of the peptide leading to neuronal apoptotic cell death. Furthermore it is also shown that Abeta can anchor itself into the membrane and suppress the neurotoxic aggregation of Abeta.

In this thesis, new methods that address some current limitations in nanoelectrospray mass spectrometry (nESI-MS) analysis are presented. One of the major objectives is the potential gain in sensitivity that can be obtained when employing the proposed techniques. In the first part of this thesis, a new emitter, based on the generation of electrospray from a spray orifice with variable size, is presented. Electrospray is generated from an open gap between the edges of two individually mounted, pointed tips. The fabrication and evaluation of two different types of such emitters is presented; an ESI emitter fabricated from polyethylene terephtalate (Paper I), and a high-precision silicon device (Paper II). Both emitters were surface-treated in a selective way for an improved wetting of the gap and to confine the sample solution into the gap. In the second part of this thesis, different methods for improved sensitivity of nESI-MS analysis have been developed. In Paper III, a method for nESI-MS analysis from discrete sample volumes down to 1.5 nL is presented, using commercially available nESI needles. When analyzing attomole amounts of analyte in such a small volume of sample, an increased sensitivity was obtained, compared to when analyzing equal amounts in conventional nESI-MS analysis. To be able to analyze smaller sample volumes, needles with a narrower orifice and a higher flow resistance were needed. This triggered the development of a new method for fabrication of fused silica nESI needles (Paper IV). The fabrication is based on melting of a fused silica capillary by means of a rotating plasma, prior to pulling the capillary into a fine tip. Using the described technique, needles with sub-micrometer orifices could be fabricated. Such needles enabled the analysis of sample volumes down to 275 pL, and a further improvement of the sensitivity was obtained. In a final project (Paper V), nESI-MS was used to study the aggregation behavior of Aβ peptides, related to Alzheimer’s disease. An immunoprecipitation followed by nESI-MS was employed. This technique was also utilized to study the selectivity of the antibodies utilized. / QC 20101112

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed. In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences. In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA). In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls. In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values &lt; 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS. In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 122-136.