A doença de Parkinson é a doença motora neurodegenerativa mais comum do mundo. Agregados proteicos contendo principalmente alfa-sinucleína é a principal marca da doença. Recentemente, tem sido demonstrado que defeitos na dinâmica mitocondrial e da autofagia são causados pelo acúmulo da proteína. No nosso estudo, foram utilizados neurônios derivados de SH-SY5Y ou de hiPSC de pacientes com a doença de Parkinson hereditária, além de leveduras para analisar a dinâmica mitocondrial e da autofagia e o envolvimento de proteínas desses processos na toxicidade da alfa- sinucleína. Foi observado a diminuição do tráfego mitocondrial em neurônios derivados das células SH-SY5Y que expressavam alfa-sinucleína A53T. Além disso a proteína mutante ainda levou ao aumento de espécies reativas de oxigênio, perturbação da autofagia e aumento da sinalização apoptótica. Os neurônios então foram tratados com NAP, um peptídeo neuroprotetor, que preveniu os efeitos tóxicos da alfa-sinucleína mutante. Leveduras contendo deleções nos genes Gem (Miro), Ypt53 (Rab5) e Atg8 (LC3) e expressando alfa-sinucleína dos tipos A30P e A53T, demonstraram que a toxicidade da alfa-sinucleína é dependente das disfunções na mitocôndria e na autofagia. A agregação da alfa-sinucleína A53T foi prevenida na ausência de Gem. Além disso, a toxicidade da proteína envolvendo a disfunção mitocondrial e sinalização apoptótica causada pelo estresse do ER foi dependente dos genes Gem e Atg8, respectivamente. Neurônios derivados de hiPSC de pacientes contendo a triplicação do gene da alfa-sinucleína, mostraram diminuição do transporte de mitocôndrias e do potencial de membrana da mitocôndria. Análises sobre a quantidade de vesículas lisossomais desses neurônios, demonstraram acúmulos dessas vesículas, sugerindo que a autofagia está alterada. Em um ensaio sobre a sensibilidade dos neurônios dopaminérgicos, foi observado que os neurônios contendo a alpha-sinucleína mutante são mais susceptíveis à rotenona, quando comparado com os neurônios dopaminérgicos do controle. A exposição à rotenona também causou mudanças na distribuição de mitocôndrias, sugerindo que o tráfego retrógrado da organela está alterado / Parkinson\'s disease (PD) is the most common motor neurodegenerative disease in the world. Protein aggregates containing mainly alpha-synuclein are a hallmark of disease. Mitochondria and autophagy defects have been suggested to be caused by alpha-synuclein toxicity. In this study, we investigated mitochondria and autophagy dynamics related to alpha-synuclein toxicity in neurons derived form SH-SY5Y cells, hiPSC from patients with familial PD or yeast. We found that SH-SY5Y neuroblastoma cells expressing A53T alfa- synuclein showed significantly inhibited mitochondrial trafficking. A53T alfa- synuclein also caused the highest increase in ROS production in the dysmobilized mitochondria in comparison to wild-type or A30P alfa- synuclein. Treatment with NAP, the 8 amino acid peptide identified as the active component of activity dependent neuroprotective protein (ADNP), completely annihilated the adverse effects of A53T on mitochondrial dynamics. During disruption of retrograde transport, we found disturbed autophagy and increased apoptosis signalization in neurons expressing A53T alpha-synuclein, suggest activation of the apoptosis pathway. Curiously, all groups expressing alpha-synuclein showed decreased levels of BCL-XL, revealing that mitochondria are susceptible to changes in the membrane potential in the presence of alphasynuclein. Nevertheless, treatment with NAP was effective in blocking the apoptosis pathway and restore autophagy. We created a model to study A30P and A53T alpha-synuclein toxicity related to Gem (Miro), Ypt53 (Rab5) and Atg8 (LC3) genes in Saccharomyces cerevisiae in which these genes were knocked down. We found that A30P alpha-synuclein toxicity was dependent on mitochondrial and autophagy dysfunction. A53T alpha-synuclein was more toxic than A30P alpha-synuclein, and its aggregation was dependent on Gem expression. A53T alpha-synuclein toxicity involving damaged mitochondrial and apoptotic signaling caused by ER stress was dependent on Gem and Atg8 genes, respectively. In a study involving dopaminergic neurons derived from hiPSCs from patients containing triplicated alpha-synuclein gene (SNCA3), we reported decreased mitochondrial trafficking and mitochondrial membrane potential, besides accumulation of lysosome vesicles. In a sensitivity assay, SNCA3 neurons demonstrated more susceptibility to rotenone toxicity, which alters intracellular mitochondrial distribution, impairing retrograde transport of the organelle

Polyglutamine (polyQ) diseases describe a group of late-onset progressive neurodegenerative disorders which are caused by the CAG triplet repeat expansion in the coding region of disease genes. Such expansions result in expanded polyQ tracts in the disease proteins which confer neurotoxicity. To date, nine such diseases are reported including Huntington's disease and several types of spinocerebellar ataxias. Misfolding of polyQ proteins and formation of intracellular SDS-insoluble protein aggregates are closely associated with the toxicity of these diseases. In particular, impairment of the ubiquitin-proteasome system (UPS) which is responsible for protein degradation has been observed in polyQ diseases. Recently, ubiquitin ligases, which govern substrate specificity of the UPS, have gained huge attention in polyQ disease pathogenesis studies. In humans, cullin (Cul) proteins, including Cul1, 2, 3, 4 & 5, are integral components of a group of ubiquitin ligases called cullin- RING ubiquitin ligases (CRLs). Each CRL displays distinct substrate specificity through specific substrate receptors. Cullin proteins are evolutionarily conserved and Cul orthologues are found in the Drosophila genome. In the present study, it was found that individual Culs displayed distinct effects on polyQ pathogenesis in Drosophila polyQ models. Particularly, it was found that Cul1 modulated polyQ-induced toxic phenotype. This modification was accompanied with an alteration in the ubiquitination level and SDS-solubility properties of expanded polyQ protein. Through genetic interaction studies and biochemical analyses, it is suggested that Cul1-based CRL specifically targets SDS-insoluble species of expanded polyQ protein for ubiquitination via selective recognition by CG2010 substrate receptor. On the other hand, it was found that expanded polyQ protein induced accumulation of CRL substrates in cells. Current data support a hypothesis that polyQ protein would impair the ubiquitin ligase activity of CRLs upon expansion of the polyQ domain, through interfering with neddylation of cullin and other uncharacterized mechanisms. Taken together, the present study identifies Cul1-CRL as a novel E3 ligase that modifies polyQ toxicity through modulating ubiquitination of expanded polyQ protein, and demonstrates a pathological mechanism by expanded polyQ protein through impairing CRL activity. These findings would lead to a better understanding of polyQ pathogenesis and give insights on developing treatments against polyQ diseases. / Wong, Kam Yan. / Adviser: Ho-Yin Edwin Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 260-273). / 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.

Polyglutamine disease is a class of neurodegenerative diseases, which is manifested by the atrophy of nervous system that results in dementia and/or motor dysfunction. The major pathological characteristics include progressive loss of neuronal cells as well as the appearance of insoluble nuclear inclusions in degenerating neuronal cells. Polyglutamine disease is caused by CAG triplet expansion in the genome. When translated, such expansion leads to the formation of expanded polyglutamine domain within the respective disease proteins and promotes abnormal protein conformational changes. Owing to their misfolded nature, the expanded polyglutamine proteins form insoluble nuclear inclusions. These insoluble nuclear inclusions are heterogeneous in nature, in which polyglutamine protein and molecular chaperones are the recruited components. All eukaryotic cells express molecular chaperones which function to mediate the proper folding of proteins. The recruitment of molecular chaperones into nuclear inclusions that contain misfolded triplet-expanded proteins strongly suggests the involvement of molecular chaperones in polyglutamine disease progression. It has been shown that over-expression of molecular chaperones in polyglutamine disease models can lead to a suppression of polyglutamine toxicity and a concomitant increase in the solubility of disease proteins, i.e. the solubility of polyglutamine disease protein is related to its toxicity. Intrigued by these observations, I aimed at elucidating the mechanism of polyglutamine disease pathogenesis by first studying the cellular regulation of endogenous chaperone expression in neurodegeneration in a transgenic Drosophila model of polyglutamine disease. A biphasic regulation of Hsp70 expression was observed, which the regulation was at the transcription level. Moreover, over-expression of Hsp70 could alter the endogenous Hsp70 protein and mRNA level of polyglutamine disease fly model. The study may help the understanding of how the chaperone expression is regulated under the effects of polyglutamine expression and thus to find out the mechanism of pathogenesis. In addition, cellular proteins that change in solubility other than disease protein will also be identified. Small heat shock proteins, glutathione S transferase and alpha 4 proteasome subunit, etc., showed change in solubility or expression by 2D gel electrophoresis analysis. Identifying the proteins that change in solubility or expression may help the finding of the interplay of proteins and thus the pathways involve in the mechanism of polyglutamine disease pathogenesis. Understanding pathogenic pathways can give ideas on how polyglutamine lead to the disease, up- or down-regulation of those protein interplays may provide direction and therapeutic candidates to suppress polyglutamine disease. / Huen Ngar Yee. / "September 2006." / Advisers: Ho Yin Chan; Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1465. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 134-146). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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One major therapeutic target for preventing and treating Alzheimer's Disease (AD) is removal of excess β-amyloid (Aβ) from the brain. Both active and passive immunotherapies targeting Aβ have proven effective in reducing brain Aβ levels and improving cognitive function in mouse transgenic models of AD. However, these approaches can induce adverse neuropathologic effects and immunologic over-activation. Indeed, clinical trials of active Aβ immunotherapy in AD patients were halted due to development of meningoencephalitis, apparently resulting from wide-spread neuroinflammation. Here we show that a more restricted and specific immune re-activation through a single adoptive transfer of Aβ-specific T cells can provide long-term benefits to APPsw+PS1 transgenic mice that last at least 1 1/2 months. Aβ-sensitive splenocytes and lymphocytes were generated in normal mice, re-stimulated with Aβ in vitro, and then adoptively transferred into cognitively-impaired APPsw+PS1 mice. Compared to control transgenic mice through 1 1/2 month post-infusion, those mice that received Aβ-sensitive T cells exhibited a reversal of pre-infusion working memory impairment and demonstrated superior basic mnemonic processing. Step-wise forward Discriminant Function Analysis of behavioral results clearly demonstrated that T cell infused mice performed comparably to wild-type non-transgenics, further emphasizing the extent of cognitive benefit this therapeutic technique afforded. Importantly, a global inflammatory response did not accompany these benefits. Though no overall reductions in Aβ deposition were noted for T cell recipient mice, a subset of T cell infused mice that benefited most in cognitive function had reduced hippocampal burdens, suggesting that hippocampal Aβ burdes did play a role in determining performance capabilities of these mice. Since chronically high levels of beta-amy loid such as those found in APPsw+PS1 mice cause immune hypo-responsive/tolerance to Aβ, our results indicate that adoptive transfer of Aβ-sensitive T-cells can supercede such immune tolerance to Aβ, and may provide a safe, long-lasting therapy for AD.

<p> <p>&nbsp / </p> </p> <p align="left">This study utilized large-scale gene expression data to define the regulatory networks of genes expressing in the hippocampus to which multiple disease pathologies may be associated. Specific aims were: ident i fy key regulatory transcription factors (TFs) responsible for observed gene expression patterns, reconstruct transcription regulatory networks, and prioritize likely TFs responsible for anatomically restricted gene expression. Most of the analysis was restricted to the CA3 sub-region of Ammon&rsquo / s horn within the hippocampus. We identified 155 core genes expressing throughout the CA3 sub-region and predicted corresponding TF binding site (TFBS) distributions. Our analysis shows plausible transcription regulatory networks for twelve clusters of co-expressed genes. We demonstrate the validity of the predictions by re-clustering genes based on TFBS distributions and found that genes tend to be correctly assigned to groups of previously identified co-expressing genes with sensitivity of 67.74% and positive predictive value of 100%. Taken together, this study represents one of the first to merge anatomical architecture, expression profiles and transcription regulatory potential on such a large scale in hippocampal sub-anatomy.</p>

Neurofilaments are the main cytoskeletal constituents in neuronal cells. They are belived to be important for maintaining the structural integrity and calibre of axons and dendrites thereby influencing the conduction velocity of nerve impulses.The neurofilament chains are divided into three groups according to their molecular size, neurofilament light (NF-L), neurofilament medium (NF-M) and neurofilament heavy (NF-H). The neurofilaments are obligate heteropolymers in vivo in which NF-L forms the backbone to which the heavier chains copolymerize to form the 10 nm neurofilament fibre. Different degenerative processes in the brain raise significant interest owing to the increasing mean age in the western world. Such diseases include amyotrophic lateral sclerosis, vascular dementia, frontal lobe dementia, progressive supra-nuclear paralysis, multiple system atrophy, low pressure hydrocephalus, and multiple sclerosis (MS). We have been able to generate six highly specific monoclonal antibodies for NF-L, and four independent epitopes were elucidated using Biacore and V8 protease degradation. Antibody 2:1 and 47:3 were selected components in a two-site ELISA assay for detection of NF-L in body fluids owing to their outstanding abililty to bind the antigen. The assay has a least detectable dose of 60 ng/l and a standard range of 60 to 64 000 ng/l. The assay was validated on its ability to detect changes of NF-L levels in CSF in patients with different neurological diseases. These were cerebral infarction, amyotrophic lateral sclerosis, relapsing remitting MS, extrapyramidal symptoms, and late onset Alzheimer’s disease. All the patient groups displayed significantly elevated NF-L levels as compared to the controls. We also tested the assay’s ability to monitor the amount of axonal breakdown in an animal model of MS. The NF-L levels were found to be elevated in rodents with chronic experimental autoimmune encephalomyelitis, giving a possible tool for monitoring new treatment strategies for axonal protection in MS. When studying a large population based MS material, we found axonal breakdown to be present early in the disease course and the breakdown was observed both in active relapse and clinically stable disease, indicative of ongoing neurodegeneration. NF-L levels were correlated to progression index, that is, high NF-L levels detected early in disease predict a fast progression of the disease. The amount of glial fibrillary acidic protein, a cytoskeletal protein found in astrocytes, was also quantified and was shown to be a good marker for the more progressive MS subtypes, that is, primary progressive and secondary progressive disease, indicating formation of astrocytic scars and activation of astrocytes. The test dealt with in this thesis has the potential to identify the slow chronic degenerative diseases with progressive disappearance of nerve cells and their large myelinated axons. There is a significant need clinically to be able to quantify such types of cell degeneration in relation to the progressive disappearance of nerve functions and to relate these different conditions to treatment regimens, disease progress, and prognosis.

L'AMPc té un paper clau com a missatger intracel.lular regulant la transmissió dels senyals extracel•lulars en diferents teixits i controlant múltiples processos cel lulars. Els nivells intracel.lulars d'AMPc es controlen mitjançant la seva síntesi, catalitzada per l'enzim adenilat ciclasa, i mitjançant la seva degradació a través de l'acció de les fosfodiesterases (PDE) de nucleòtids cíclics. Hi ha 11 famílies de PDEs. La PDE4 representa a una família de fosfodiesterases específiques d'AMPc formada per quatre gens paràlegs (PDE4-D), cadascun dels quals és capaç de generar múltiples variants d’splicing. La PDE4A, la PDE4B i la PDE4D es troben expressades en diferents tipus de cèl lules inflamatòries on tenen un important paper com reguladores dels processos inflamatoris. La inhibició selectiva, tant in vitro com in vivo, de PDEs ha demostrat tenir diferents efectes antiinflamatoris. En aquest treball es mostra la implicació de la isoforma PDE4B, i en concret la seva variant de splicing PDE4B2, durant el procés neuroinflamatori del model animal d’Esclerosis Múltiple, l’Encefalomielitis Autoimmune Experimental (d’EAE). Els resultats mostren un augment de l’expressió de l’ARNm de PDE4B2 a la medul•la espinal de ratolins EAE que correlaciona amb l’expressió d’alguns marcadors inflamatoris de forma dependent a la simptomatologia clínica dels animals. També s’observa que l’enzim PDE4B es trova localitzat principalment en cèl•lules presentadores d’antigen (APCs) com les cèl•lules dendrítques i els macròfags/micròglia. A més, els ratolins PDE4B-/- mostren una aparició temprana dels símptomes clínics en comparació amb els ratolins wildtype, amb alteracions en l’expressió de l’ARNm d’algunes citocines. L’alteració selectiva de la PDE4B2 en el model d’EAE en ratolí i la seva participació en el desenvolupament de la malaltia com s’ha observat en els animals PDE4B-/- presenta noves possibilitats sobre l’ús d’inhibidors selectius per les diverses isoformes (i variants d’splicing) tant per aplicacions terapèutiques com per investigar mecanismes d’inflamació en malalties neurodegeneratives. / Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that courses with neuroinflammation, axonal damage and demyelination. The model is characterized by T- and B-cell responses to myelin oligodendrocyte glycoprotein which produce a wide range of pro- and anti-inflammatory cytokines. The modulation of cAMP levels through pharmacological manipulation of phosphodiesterases (PDEs) provokes profound anti-inflammatory responses. In the EAE model, amelioration of the clinical signs and delayed onset is observed after PDE4 inhibition and the PDE4B gene has been related to the inflammatory immune response in mice. Here we analyzed post-immunization changes in the expression of mRNA coding for the PDE4B2 splice variant by semiquantitative real-time PCR and in situ hybridization. The results showed an upregulation of PDE4B2 mRNA in the spinal cord of EAE mice which correlates with FoxP3 and TGF-β mRNAs expression in a score-dependent manner. We also found that PDE4B enzyme is mainly localized in antigen-presenting cells (APCs) such as dendritic cells and microglia/macrophages. PDE4B-/- mice show an earlier onset of the disease compared to wildtype mice, with alterations in some cytokine mRNA expression. The results point to a protective role of the PDE4B enzyme and PDE4B2 splice variant in particular, during EAE pathogenesis by modulating cAMP levels in APCs and controlling the cytokine environment for T-cell differentiation.

The nervous and cardiovascular system plays the most complex and vital role in all organisms. Any damage or injury to these essential organs in our body results in long term irreversible impairment or death. The main goal of the regenerative medicine is to repair or recreate tissues using stem cells to restore the vital function of the targeted organ. Along with organ specific stem/progenitor cells, non-toxic, biodegradable synthetic polymers are also needed for an effective reparative therapy. The effect of PCL materials and surface modified (PEDOT coated) PCL materials of different topology with neural progenitor cells as test platforms are evaluated for cytotoxicity and neuron differentiation. The stem cells from heart are isolated and characterized as cardiac stem cells by Fluorescence activated cell sorting through specific antigen expression. The cardiac stem cells are used to establish effective proliferation and differentiation system. Hence, developing cardiac and neural progenitor cell cultures as an efficacy test platforms for biomaterials of different diameter and orientation benefits respective tissue engineering with proper restoration of function. Further, the nerve and cardiac tissue rejuvenation would serve as a regenerative therapy for numerous neurodegenerative disorders and cardiovascular disorders like myocardial infarction respectively.

Retrospective studies suggest that lifestyle activities may provide protection against Alzheimer s Disease (AD). However, such studies can be inaccurate and prospective longitudinal studies investigating lifestyle protection against AD are both impractical and impossible to control for. Transgenic (Tg+) AD mice offer a model in a well controlled environment for testing the potential for environmental factors to impact AD development. In an initial study, Tg+ and non-transgenic (Tg-) mice were housed in either environmentally enriched (EE) or standard housing (SH) from 2-6 months of age, with a behavioral battery given during the last 5 weeks of housing. In the Morris maze, platform recognition, and radial arm water maze tasks, Tg+/EE mice were completely protected from cognitive impairment present in Tg+/SH mice and comparable to control Tg-/SH mice in cognitive performance. The current study utilized the same cognitive-based behavioral battery and multimetric statis statistical analysis to investigate the protective effects of "complete" environment enrichment (EE) versus several of its components (physical activity, social interactions) in AD transgenic mice. The AD transgenic mice utilized develop beta-amyloid (AB) deposition and cognitive impairment by 6-7 months of age. Similar to our initial study, results show that "complete" EE (physical, social, and cognitive activities) from 2 to 8 months of age completely protected AD transgenic mice from cognitive impairment in tasks representing different cognitive domains - working memory, reference learning, and search/recognition. In strong contrast, Tg+ mice reared in environments that included physical activity and social interaction, or only social interaction, were not protected from cognitive impairment in adulthood -- enhanced cognitive activity was required over and above that present in these other environments. Through use of discriminant function analysis, EE and/or NT mice were consistently discriminated from the poorer performing other housing groups. The cognitive benefits observed in EE-housed Tg+ mice occurred without significant changes in cortical AB levels, plasma cytokine levels, or plasma corticosterone levels, suggesting involvement of mechanisms independent of these endpoints. However, EE-housed Tg+ mice did have decreased dendritic length of neurons in the parietal cortex (but not hippocampus). Noteworthy is that plasma cytokine levels and hippocampal dendritic length consistently correlated with cognitive measures, suggesting their involvement in underlying mechanisms of cognitive performance. The present work provides the first evidence that "complete" EE (including enhanced cognitive activity) is needed to provide cognitive protection against AD in a Tg+ model of the disease, while the physical and social activity components of EE do not alone lead to protection. These results suggest that humans desiring to gain maximal environmental protection against AD should live a lifestyle high in cognitive, social, and physical activities together.