Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Neurogenesis is the process by which functionally integrated neurons are generated from progenitor cells. In the adult mammalian brain two sites of high density cell division have been identified that contain neural progenitor cells retaining the ability to generate new neurons: the subgranular zone of the hippocampus (SGZ) and the subventricular zone (SVZ) lining the lateral ventricles in the forebrain. Several studies have suggested that SVZ neural progenitor cells in the adult brain can migrate into regions other than the olfactory bulb after either administration of growth factors, induction of neuronal cell loss or injury. Brain-derived neurotrophic factor (BDNF) and fibroblast growth factor (FGF-2) play major roles in regulating the survival and fate of progenitor cells in the adult mammalian brain. To determine the effect of BDNF or FGF-2 on neurogenesis in the injured adult brain, BDNF or FGF-2 were over-expressed in the subventricular zone (SVZ) via recombinant adeno-associated virus (AAV1/2) delivery and newly generated cells were identified using bromodeoxyuridine (BrdU; 150mg/kg intraperitoneal) labelling. Selective striatal cell loss was induced in a subgroup of rats by unilateral striatal injection of the excitotoxin quinolinic acid (QA) 21 days after AAV1/2 injection and 24 hours prior to BrdU labeling. The results of this thesis demonstrate that BDNF augments the recruitment, neuronal differentiation and survival of progenitor cells in both neurogenic and non-neurogenic regions of the unlesioned or QA lesioned brain. BDNF also appears to contribute to the persistence of newly generated neurons in the QA lesioned striatum. Our results provide the first evidence demonstrating the neurogenic effect of BDNF on compensatory striatal neurogenesis in the injured adult brain and suggest that enhanced BDNF expression may be a viable strategy for inducing or augmenting endogenous neural progenitor cell neurogenesis. Unlike the effect of BDNF, FGF-2 appears to have no effect on proliferation and/or survival of neural progenitor cells in either the normal or damaged brain. FGF-2 appears to be unable to act as a positive mediator of SVZ progenitor cell proliferation and neurogenesis in this study. However, FGF-2 may be having an inhibitory effect on progenitor cell differentiation. The negative result of the FGF-2 study may be of major significance in indicating the potential requirement of additional factors interacting with FGF-2 to influence neurogenesis. The results from the FGF-2 study contribute to the research field in highlighting the complexity of the mammalian neurogenic process. This thesis highlights the need for further investigation into multiple factor interactions, tighter regulation of the transgenic protein expression from the AAV1/2 delivery vector or alternative progenitor cell labelling paradigms. However, it does show that if neurogenesis can be induced or augmented exogenously, neural progenitor cells may provide a substrate for repair in the adult brain and dramatically change therapeutic approaches towards the treatment of neurodegenerative diseases.

Orientador: Iscia Lopes Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T07:28:20Z (GMT). No. of bitstreams: 1 Paiva_RejaneScolariRezende_D.pdf: 1128972 bytes, checksum: 7861376a51c6236c8ecffe342b18ce56 (MD5) Previous issue date: 2006 / Resumo: Com o avanço da genética molecular, hoje é possível detectar alterações genéticas específicas responsáveis por várias doenças, até mesmo antes de o indivíduo manifestar qualquer sintoma. Assim, testes genéticos podem ser realizados para a confirmação diagnóstica, o teste diagnóstico (TD) e para a realização de testes pré-sintomáticos ou teste preditivo (TP). Neste trabalho abordamos questões relacionadas ao TP, para dois grupos de doenças neurodegenerativas: a doença de Huntington (DH) e as ataxias espinocerebelares (AEC). Ambas são doenças degenerativas, progressivas, geralmente familiares, cujos sintomas se iniciam em idade variada entre a primeira e a quinta década de vida, progridem lentamente, levando ao óbito após 15 a 20 anos. Nossos objetivos principais foram: a) determinar o perfil psicológico dos usuários do TP, b) conhecer as motivações que levam os indivíduos a realizarem o teste preditivo, c) avaliar psicologicamente estes indivíduos para determinar se os mesmos se encontram preparados para se submeterem ao teste molecular preditivo. Procuraram o serviço para a realização do TP 38 indivíduos que tinham a confirmação molecular de um membro da família afetado pela DH ou AECs. A maioria (n=23) dos indivíduos interessados no TP, estavam em risco para a doença de Huntington e 40% (n=15) para ataxia. Sessenta e seis por cento (n=10) dos indivíduos em risco para ataxia tem história familiar e confirmação molecular para a Doença de Machado-Joseph. Destes 38 indivíduos que procuraram o serviço, apenas 34% (n=13) concluíram o protocolo do TP e 50% (n=19) interromperam o mesmo. Para 16% (n=6) dos indivíduos que requisitaram uma consulta, o interesse foi obter informações referentes ao risco de transmissão, bem como a conduta para a realização do teste. O objetivo dos usuários em realizar o TP foi para 38% (n=5), o planejamento familiar. Para 32% (n=4) receber o resultado do teste foi importante para reduzir a incerteza sobre ser portador da mutação e assim poder planejar o futuro sem a preocupação de desenvolver a doença neurodegenerativa presente em toda sua vida. Já para 23% (n=3) dos usuários do TP, a grande motivação foi querer descartar o risco para a prole e para 7% (n=1) o planejamento profissional foi questão a ser resolvida após o resultado do TP. Com este trabalho pudemos concluir que os indivíduos que realizaram o TP estão com a vida afetiva, profissional e financeira estável. Além disso, não houve diferença nos resultados no que diz respeito às condições físicas e emocionais destes usuários que se submeteram a avaliação depois de conhecerem o resultado / Abstract: Recent advances in molecular genetics have made it possible to detect specific genetic alterations that are responsible for several diseases, even before the individual presents any symptoms. Hence, genetic tests such as the diagnostic test (DT) to confirm diagnosis and the predictive test (PT) or presymptomatic test can be performed. This study deals with the PT aspects of two neurodegenerative diseases: Huntington¿s disease (HD) and spinocerebellar ataxias (SCA). Both are degenerative, progressive, generally familial diseases with onset of symptoms between the first and fifth decade of life, progressing slowly to death after 15 to 20 years. Our main objectives were: a) determine the psychological profile of PT users; b) study the reasons that lead individuals to undergo predictive testing; c) psychological assessment of these individuals to check if they are prepared to undergo molecular testing. Thirty-eight individuals having a family member affected by HD or SCA came to this service for testing. Their ages ranged from 17 to 53 years, their educational levels were middle school, high school and university, 58% (22) were females. More than half of the individuals (n=23) interested in undergoing the PT presented a risk for Huntington¿s disease and 40% (n=15) for ataxia. Sixty percent (n=10) of the individuals with a risk for ataxia had molecular testing confirmation for Machado- Joseph Disease. Only 34% (n=13) of the 38 individuals concluded the PT protocol, while 50% (n=19) did not. Sixteen percent (n=06) of the individuals who requested a consultation were only interested in information about risk of transmission and the test procedure. The test users presented a 38% risk for Huntington¿s disease and 62% (n=8) risk for ataxia. Fifty percent of the ataxia test users presented a risk for type 3 or Machado-Joseph disease. In the case of 38% (n=5) of the users, their main purpose in performing the PT test was family planning. However, one individual already had a child, but wanted to plan a second child if he was not a carrier of the mutation responsible for the disease in his family. The test results were important for 32% (n=4) of these individuals because they cleared doubts about being mutation carriers, which meant they could plan the future without worrying about developing a neurodegenerative disease. In the case of 23% (n=3) PT users, the main reason was to discard any risk in relation to their offspring and in the case of 7% (n=1), their professional planning could be done based on the results. Hence, we can conclude that an organized protocol offering genetic counseling and pre-test services is needed so that individuals who would like to undergo the PT are able to evaluate the consequences of this test and their reasons for performing the test / Doutorado / Genetica Medica / Doutor em Ciências Médicas

Oligodendrocytes (OLs) are the cells responsible for myelin production in the central nervous system (CNS). Myelin serves to increase the efficiency of signal propagation down the axon and is essential for proper communication between the CNS and the periphery. As a result, pathologies affecting the OL, including multiple sclerosis (MS) and multiple system atrophy (MSA), present with a wide range of symptoms including impaired muscle control, loss of coordination, as well as cognitive deficits. While the biology of the OL continues to garner research interest, much remains to be understood about cell function in a healthy context, and also how the biology of these cells goes awry in disease. Our objective was to explore the effects of varying disease models on OL biology and use those findings to further our knowledge on the biology of OL development and regeneration. Here we explore OL function and dysfunction in the context of spinal muscular atrophy (SMA), MSA and MS. We have thoroughly characterized the OL response to SMN-depletion and have determined that SMN is not required for the development of OLs in the neonatal brain. Additionally, we have sought to characterize the endogenous role of MSA-disease relevant protein alpha-synuclein in OL development and have determined that this protein is not required for OL differentiation or CNS myelination. Lastly, we have explored the biology of the OL in the context of the inhibitory milieu it faces during remyelination in MS. We have investigated different pathways that may be involved in mediating signalling of one such inhibitory cue (chondroitin sulphate proteoglycans, CSPGs), and have extended this model to interrogate OL cytoskeletal dynamics in the context of CSPGs. Together, this work uses disease frameworks to investigate basic OL biology, as well as provides insights into how the OL and its interactions with the extracellular milieu should be considered in disease pathogenesis and therapeutic exploration.

The etiology of most neurodegenerative diseases remains a mystery. Environmental factors seemingly play an important role in neurodegenerative diseases, as does the immune system. Here, we describe immunological phenotypes associated with the neurodegenerative diseases AOA2 and ALS. During CSR, B-cells and neurons share a preferred pathway for DNA repair, NHEJ. SETX, the gene implicated in AOA2, has been implicated in the DDR. In SETX-deficient conditions, B-cells exhibit a defect in IgA class-switching as a result of impaired NHEJ and enhanced alternate-end joining, a slower process with higher mutational burden. These results suggest that neurons in SETX-deficient patients may also demonstrate defects in DNA repair, leaving them more susceptible to death. Further, IgA plays an important role in both protection from infections and as an anti-inflammatory mediator at mucosal surfaces. Defects in IgA class switching might lead to immune dysregulation in AOA2, similar to that observed in other neurodegenerative diseases. Viral infections have been associated with several neurodegenerative diseases, including ALS. However, a causal role for viruses in the etiology of ALS has never been established. Common viral infections can impact immune cell phenotypes within the CNS. Microglia are the primary immune cell of the CNS and are able to respond to subtle changes in the microenvironment. Microglia have neurotoxic properties upon hyper-activation. Influenza infection of SOD1G93A mice accelerated ALS disease progression and reduced overall survival. Exacerbated microgliosis was evident within the spinal cords of infected mice. Thus, the immune response stimulated by viral infections may result in toxic microgliosis that accelerates the progression of ALS. Together, this body of work describes a series of novel immunological abnormalities associated with AOA2 and ALS4. A deeper understanding of the role played by the immune system in patients with neurodegenerative disorders may unveil new targets for future therapies. / Thesis / Doctor of Philosophy (PhD) / The causes of most neurodegenerative diseases remain poorly understood. These diseases are highly complex, as they are influenced by both genetics and the environment and involve many different cell types. However, neurodegenerative diseases also share common hallmarks. The immune system is known to behave abnormally in several neurodegenerative diseases. Therefore, we set out to study the involvement of the immune system in two related neurodegenerative diseases, ataxia with oculomotor apraxia type 2 (AOA2) and amyotrophic lateral sclerosis (ALS). We demonstrate that AOA2-like mutations in senataxin (SETX) affect the formation of specific antibodies due to problems repairing broken DNA. We also find that viral infections activate immune cells within the spinal cord which can promote ALS progression. Taken together, this body of work suggests that the immune system plays an important role in AOA2 and ALS, and that drugs that prevent abnormal immune responses might help to treat these diseases.

Brain inflammation has been implicated with the pathogenesis of neurodegenerative diseases. Activated microglia and endothelial cells induce production of reactive oxygen species (ROS) and overexpress pro-inflammatory mediators that perpetuate tissue damage. Current treatments are not effective against progressive stages of neurodegenerative diseases and more advanced therapies need to be developed. Recently, nanomaterials have been investigated for therapeutic applications. Nanoparticles can increase efficiency of drug delivery due to increased tissue distribution and the ability to modify surface chemistry to increase biocompatibility and incorporate targeting moieties. In the present study, we established in vitro and in vivo brain inflammation models by administering lipopolysaccharide to mouse brain endothelial cells, microglia, macrophage cells and C57BL/6 male mice. Changes in mRNA expression of pro-inflammatory mediators were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), E-selectin, and intercellular adhesion molecule-1 (ICAM-1) displayed significant overexpression when compared to the control. Additionally, folate receptor-α (FR-α) was also overexpressed, confirming that our model will function appropriately for specific targeting experiments. Cellulose nanocrystals are rod-like particles, approximately 5 nm wide and 100-150 nm long. The surface area consists of extended hydroxyl groups and the structure is hydrophilic in nature. These characteristics make cellulose nanocrystals ideal for surface modification and ensuring long blood circulation half-life. Cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] conversion assay and a Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit. At each concentration of cellulose nanocrystals (10, 25, 50 μg/mL), both assays showed the nanoparticles to be non-toxic. Binding/uptake experiments utilizing a fluorescence plate reader and fluorescence microscope showed no non-specific uptake of untargeted cellulose nanocrystals. In contrast, when conjugated to folic acid, cellulose nanocrystals were selectively incorporated to folate receptor-overexpressing cells. These results indicate that both in vitro and in vivo brain inflammation models can be utilized to assess therapeutic efficacy of folate receptor-targeted bioconjugated nanoparticles. / Master of Science

Magister Pharmaceuticae - MPharm / Neurodegenerative diseases are multifactorial in nature, and taking the complex nature of these disorders into consideration, multi-target directed ligands may present as better options to treat these disorders than the classic ‘one molecule, one target’ approach. Neurotransmitter amines are catabolized by monoamine oxidase A and B (MAO-A and MAO-B), therefore they have been targeted for the treatment of neuropsychiatric and neurodegenerative diseases. Besides offering a potential antidepressant action in PD, MAO-A inhibitors may also provide a symptomatic benefit by reducing MAO-A-catalysed oxidation of dopamine. The oxidative deamination reaction catalyzed by MAO-B is one of the major catabolic pathways of dopamine in the brain. Inhibition of this enzyme leads to enhanced dopaminergic neurotransmission and are currently used in the symptomatic treatment of PD. Furthermore, MAO-B inhibitors may also exert neuroprotective effects by reducing the concentration of potentially hazardous by-products produced by MAO-B-catalysed dopamine oxidation. Apoptosis or programmed cell death occurs in a number of neurodegenerative disorders and it has been proven that inhibition of the executing enzyme, caspases-3, slows down or even stops apoptosis. Having this in mind we focused on the propargylamine function of selegiline and the fluorophenyl function of safinamide, because of their inherent monoamine oxidase inhibitory activities; and isatin as a non-peptide inhibitor of caspase-3. Therefore we attempted to design and synthesize multifunctional hybrid molecules acting simultaneously to halt the apoptotic neuronal breakdown process and eliminate the signs and symptoms of diseases such as PD. Seven novel compounds were successfully synthesized utilizing multistep processes. The synthesis of 5 chlorosulfonyl isatin was accomplished starting from the commercially available isatin in two steps, which were, sulfonation using tetramethylene sulfone and chlorination with POCl₃. Next 5-chlorosulfonyl isatin was conjugated to the fluorophenylamine derivative with the fluoro-function at either the ortho, meta or para position through a nucleophilic substitution reaction on the chlorosulfonyl position. The resultant compounds were coupled on the N position of the isatin function with propargyl bromide, using a microwave synthesis procedure, in a nucleophilic substitution reaction. The structures and purity were confirmed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). In the biological evaluations recombinant human MAO-A, MAO-B and caspase 3 enzymatic assays were performed to determine the activity of the novel compounds at an enzymatic level. The inhibition percentages for these compounds were calculated and plotted against the logarithm 8 of the inhibitor concentrations to obtain a sigmoidal dose-response curve and consequently the IC50 value. The synthesized compounds showed inhibition of the MAO-A, MAO-B and caspase-3 enzymes at low to high micro molar concentrations. The role of the fluorophenylamine moiety in the synthesized compounds was significant for their multifunctional activity as shown by compounds 3 and 4 having good inhibitory activity towards MAO-A, MAO-B and also excellent inhibitory activity against caspase 3, making those ideal candidates for further lead compound development and multifunctional drug design. The introduction of the propargylamine moiety only increased the MAO-A inhibitory activity; this was shown by compounds 7, 8 and 9 which exhibit good MAO-A selectivity with low MAO-B and caspase-3 inhibitory activities.

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