MITOCHONDRIAL GENE EXPRESSION IN HUMAN MONONUCLEAR CELLS By Monika D. Ruchała, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014. Director: Dr. James P. Bennett Jr, M.D., Ph.D., Bemiss Professor Departments of Neurology, Psychiatry and Physiology and Biophysics Adult neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), have been intensively studied in recent years in pursuit of mechanisms responsible for origin and progression. One emerging theme is mitochondrial energetic deficiency as a mechanism of neuronal death. Recent descriptions of protocols to generate induced pluripotent stems cells (iPSCs) from living patients offer the potential to create unique disease models. This model can potentially lead to crucial advances in developing treatment options for a wide variety of neurodegenerative diseases. In this thesis, we attempt to induce iPSCs from mononuclear cells (MNC) in peripheral blood acquired from patients with ALS and healthy control (CTL) subjects, and analyze their mitochondrial genomes. The reprogramming of MNC to yield iPSC was done by nucleofection of an episomal plasmid pEB­ C5, expressing OriP sequences of the Epstein­Barr and five reprogramming transgenes Oct4, Sox2, Klf4, c­Myc and Lin28. We investigated the expression of mitochondrial DNA genes, ND2, ND4, COXIII and 12s rRNA in the ALS and CTL MNC before and after their culturing. The results implicate deregulated mitochondrial bioenergetics as a characteristic of ALS. Future work will establish whether these abnormalities in mitochondrial bioenergetics persist in iPSC’s and iPSC-derived neurons from ALS subject

La Sclérose Latérale Amyotrophique se caractérise par la perte sélective de motoneurones (MNs) du cortex, du tronc cérébral et de la moelle épinière. Les souris surexprimant le gène humain muté codant pour la superoxide dismutase 1 (mSOD1) constitue un bon modèle d'étude. Les MNs mSOD1 présentent une hypersensibilité à la mort après activation du récepteur Fas et la production d'oxyde nitrique (NO). Notre étude protéomique a identifié deux effecteurs du NO, la calréticuline (CRT) et CRMP4. CRT est une protéine chaperonne de stockage du calcium dans le réticulum endoplasmique. Nous montrons que, in vivo, CRT diminue de moitié dans une sous population de MNs mSOD1 dits vulnérables, car dégénérant les premiers. Sa diminution est nécessaire et suffisante pour induire la mort des MNs mSOD1 en activant le stress du RE. CRMP4 est une protéine de régulation de la croissance axonale, qui augmente in vivo dans les MNs mSOD1 à un stade pré-symptomatique. Sa surexpression est suffisante pour induire une dénervation périphérique et la dégénérescence de MNs mSOD1. Nos résultats mettent en évidence CRT et CRMP4 comme étant deux cibles thérapeutiques potentielles dans la SLA. / Amyotrophic Lateral Sclerosis (ALS) is characterized by the selective degeneration of upper and lower motoneurons (MNs). The most common familial form and best characterized mouse model of ALS is linked to mutations in the gene coding for the superoxide dismutase 1 (mSOD1). MNs expressing mSOD1 show an increased sensitivity to the death induced by Fas/NO activation. Our proteomic study identified two downstream effectors of NO, Calreticulin (CRT) and CRMP4. CRT is a chaperone-calcium-binding protein of the endoplasmic reticulum, which is decreased two-fold in vivo, in an early degenerating MNs sub-population, named vulnerable. The decrease in CRT expression is both necessary and sufficient to kill mSOD1 MNs through ER stress activation. CRMP4 is a neurite outgrowth regulator which expression is increased in vivo in mSOD1 MNs at a presymptomatic stage. CRMP4 overexpression is sufficient to induce peripheral denervation and, through a dying-back effect, to kill mSOD1 MNs. Our results point out CRT and CRMP-4 as two potential therapeutic targets for ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism. Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur.  Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.

La nanobiomécanique est un domaine interdisciplinaire émergent qui apporte une contribution significative à l'étude des processus biologiques liés à des diverses maladies humaines. Dans le cadre de ces travaux, des méthodes nanomécaniques basées sur la microscopie à force atomique ont été directement appliquées pour comprendre les processus pathogènes sous-jacents de deux maladies qui sont la formation de métastases cérébrales et à la sclérose latérale amyotrophique.Une cellule tumorale maligne, pour se propager et former des métastases, doit modifier et adapter en permanence ses propriétés adhésives et élastiques. Ici, nous avons combiné la cartographie des forces avec la spectroscopie de force monocellulaire (SCFS) afin d'obtenir un aperçu direct du processus de "criblage" de surface des cellules tumorales pendant leur extravasation dans le parenchyme cérébral. Les cartes élastiques et adhésives obtenues ont montré que les propriétés adhésives ne dépendent que faiblement des caractéristiques élastiques, et souligne l'importance des liaisons à long distances de type attache pour des adhésions réussies.Afin d'étudier comment le potentiel métastatique est lié aux propriétés nanomécaniques des cellules tumorales, des mesures SCFS comparatives ont été effectuées entre trois types de cellules mélanomateuses (WM35, A2058 et A375), montrant des caractéristiques invasives altérées. Nos résultats indiquent une faible élasticité relative, une adhérence maximale élevée et un nombre de liaisons individuelles élevé, comme propriétés clés des cellules mélanomes hautement métastatiques. En conclusion nos études permettent de catégoriser les cellules suivant leur potentiel métastatique agrandissant dans l’ordre suivant : WM35, A2058 et A375.La pathogenèse de la sclérose latérale amyotrophique (SLA) est multifactorielle et ce caractérise également par la présence de processus inflammatoires. Nos études montrent des différences significatives dans l'adhésion de cellules T cytotoxiques CD8+ isolées chez des souris mutantes SOD1G93A et ce de type sauvage avec des neurones moteurs. Les cellules T dérivées de souris mutantes présentent une force d'adhésion améliorée par rapport aux souris saines dérivées. En outre, elle présente une réduction très significative après le blocage des liaisons entre pMHC-I et TCR. Ces résultats corroborent la contribution des lymphocytes T cytotoxiques au développement de la SLA, en tant qu'acteur actif de la neurodégénérescence.Les myoblastes jouent un rôle primaire dans les processus moléculaires impliqués dans le développement musculaire, le vieillissement et la réparation. Les cartes nanomécaniques à haute résolution réalisées sur des myoblastes et des myotubes multi-nucléaires, isolés à partir d'un modèle de souris SLA, ont révélé des modifications d'élasticité lors de la différenciation des myotubes. Des augmentations significatives du module d'élasticité ont été observées dans les projections des myoblastes allongés par rapport à leur corps cellulaire. Pour les myotubes, des différences ont été observées entre l'élasticité d'une population mince et épaisse de myotubes de souris sain. Cependant, chez le mutant SOD1, l'augmentation observée du module élastique de la population mince suggère un durcissement autonome accru des myotubes dérivés de souris SLA.En résumé, nos études mécaniques au niveau cellulaire ont décrypté divers aspects de deux pathologies graves, ce qui a donné lieu à de nouvelles découvertes sur leurs processus fondamentaux. Ces travaux mettent en lumière la pertinence et adéquation des méthodes d’études nanomécaniques basées sur l’AFM pour révéler des connaissances précieuses sur la physiopathologie, le développement, le diagnostic et la progression des maladies. / Nano-biomechanics is an emerging field of science that opened a new horizon in scientific research by generating significant contribution in the study of human diseases. In this work, atomic force microscopy-based nanomechanical methods were directly applied in order to elucidate important questions related to brain metastasis formation and amyotrophic lateral sclerosis (ALS). Better understanding of the underlying pathogenic processes of these life-threatening diseases is fundamental for the advancement of early diagnostics and improved therapeutics.A malignant tumor cell, in order to spread and form metastasis, has to vary and continuously adapt its adhesive and elastic properties. Here, we combined force mapping with single cell force spectroscopy (SCFS) in order to gain direct insight into the surface “screening” process of tumor cells during their extravasation into the brain parenchyma. Intercellular adhesive forces and works, as well as elastic properties were spatially mapped showing that adhesive properties are only slightly dependent on elastic characteristics, and highlighting the importance of long range tether-like linkages for successful adhesions.In order to examine how the metastatic potential relates to tumor cell’s autonomous and inter-cellular nanomechanical properties, comparative SCFS measurements were performed between three melanoma cell types (WM35, A2058 and A375), showing altered invasive characteristics, and blood vessel lining endothelials. Our results indicate low relative elasticity, high maximal adhesion and high number of individual linkages, as the key properties of highly metastatic melanoma cells. This enables us to suggest the following ordering of tumor cells from lower to higher metastatic potential: WM35, A2058 and A375.The pathogenesis of ALS is multifactorial, being characterized by the presence of inflammatory processes as well. In our results significant differences have been observed, by comparing the adhesion of CD8+ cytotoxic T cells isolated from wild-type as well as SOD1G93A mutant mice against wild-type motor neurons. Mutant mice derived T cells show enhanced adhesion strength, compared to the healthy mice derived ones. Moreover, we observe a significant reduction of T cell - motoneuron interactions after blocking the specific recognition bindings between pMHC-I and TCR. These results corroborate the contribution of cytotoxic T cells in the development of ALS, as an active player in neurodegeneration.Investigating primary myoblasts allow deciphering molecular processes involved in muscle development, aging and repair. Therefore, in our study high resolution nanomechanical mapping was performed on single elongated myoblasts and multinuclear myotubes, isolated from an ALS mouse model, to reveal elasticity features during early differentiation stage into myotubes. We have found that projections of the elongated myoblasts show significantly increased elastic modulus values compared to their cell body. Regarding myotubes, differences have been observed between the elasticity of a thin and thick population of wild-type myotubes, indicating the different maturity of the two populations. However, in SOD1 mutant, the observed increase in the elastic modulus of the thin population suggests an enhanced autonomous hardening of ALS derived myotubes.In summary, our cellular-level mechanical studies deciphered various aspects of two different life-threatening pathologies resulting in novel discoveries about their fundamental processes. This work highlights the high impact and the important role of AFM-based nanomechanical methods in providing valuable knowledge about disease pathophysiology, development, diagnostics and progression that could contribute to the evolvement of future therapies.

Bakgrund: Amyotrofisk lateralskleros är en neurologisk sjukdom som orsakar muskelförtvining. I Sverige insjuknar årligen runt 220–250 personer i ALS och totalt lever cirka 750–850 personer med sjukdomen. Etiologin är ännu okänd för sjukdomen som leder till döden.   Syfte: Att belysa upplevelsen av att vara närstående till personer med ALS.   Metod: En kvalitativ intervjustudie baserad på sex intervjuer vilka analyserades utifrån en kvalitativ manifest innehållsanalys.   Resultat: I analysprocessen framkom tre huvudkategorier; Närståendes upplevelser att leva med personer med ALS, Närstående upplevelser av vården och Närståendes önskemål angående vårdmöten. Att leva i den direkta närheten av någon som drabbats av ALS skapar mycket oro, ångest och förtvivlan. Närstående upplever förtvivlan då informationen anses otillräcklig vilket också gäller kunskapen kring sjukdomen bland vårdpersonalen.   Slutsats: Att drabbas av ALS är att få sin dödsdom. Sjukdomen drabbar inte bara patienten utan också närstående. Den slutsats som kan dras utifrån studiens resultat är att det saknas kunskap, information och stöttning av de närstående.

A Esclerose Lateral Amiotrófica (ELA), também conhecida como Doença de Lou Gehrig, é a forma mais comum de doença do neurônio motor. Tem início geralmente tardio (4ª/5ª década de vida), afetando tanto os neurônios motores superiores quanto os inferiores. A degeneração provocada pela ELA é progressiva e irreversível. Em geral, a evolução da doença é rápida, levando os pacientes ao óbito entre 3 e 5 anos após o início dos sintomas, devido principalmente à falência respiratória. Atualmente, o único medicamento liberado pelo FDA (Food and Drug Administration) para o uso em ELA é o Riluzol, que tem um efeito mínimo na expectativa de vida dos pacientes. Neste cenário, a terapia celular vem sendo avaliada como uma possível alternativa. Estudos pré-clínicos indicam efeitos benéficos do tratamento de camundongos SOD1 (modelo animal para ELA) com células estromais mesenquimais ou simplesmente células mesenquimais (MSCs), atribuída principalmente à ação de fatores solúveis. Aqui propusemos o uso de pericitos, uma linhagem celular ainda não testada para tratamento pré-clinico em modelo murinho de ELA. Pericitos são células perivasculares que circundam células endoteliais e que desempenham importantes funções celulares como por exemplo participação da formação e manutenção da barreira hematoencefálica, essencial para proteger o sistema nervoso central de danos em doenças neurodegenerativas. Dessa forma, este trabalho pretendeu comparar o potencial terapêutico de células mesenquimais e pericitos obtidos do tecido adiposo humano de um mesmo doador, em camundongos SOD1. Para tal, testes físicos (peso, PaGE, motor score, rotarod) foram aplicados semanalmente e a sobrevida dos animais foi avaliada. Os resultados demonstram que, com exceção dos benefícios observados nos testes do PaGE e do motor score em uma fase mais inicial da doença, o tratamento com MSCs ou pericitos não resulta em efeitos significativos no quadro clínico de camundongos SOD1 do sexo feminino. Para os machos, o tratamento com pericitos se destaca em relação aos tratamentos com MSCs ou HBSS (veículo), resultando em efeitos benéficos na sobrevida e em determinadas funções motoras dos animais, com destaque para os testes do motor score e do rotarod, onde há uma melhora na fase inicial da doença. A análise da expressão gênica no cérebro e na medula de animais em fase final da doença sugere que o tratamento de machos com pericitos é capaz de estimular as defesas antioxidantes do animal. Ainda nestes órgãos, não foram encontrados vestígios das células humanas injetadas, indicando um possível efeito sistêmico das mesmas / Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig\'s disease, is the most common form of motor neuron disease. Most cases are characterized by an adult onset of symptoms, usually in the fourth or fifth decade of life, affecting both upper and lower motor neurons. The degeneration caused by ALS is progressive and irreversible. On average, the survival ranges from 3 to 5 years after onset, mainly due to respiratory failure. Currently, the only Food and Drug Administration (FDA)-approved medication for this disorder is Riluzole, but its effects on survival are minimal. In this scenario, cell therapy is being evaluated as a possible alternative. Preclinical studies indicate beneficial effects of treatment of SOD1 mice (animal model for ALS) with mesenchymal stromal cells or simply mesenchymal cells (MSCs), mainly attributed to the action of soluble factors. Here we propose the use of pericytes, a cell line not yet tested for preclinical treatment in of ALS. Pericytes are perivascular cells surrounding endothelial cells and play important cellular roles such as assistance of formation and maintenance of the blood-brain barrier, which is essential to protect the central nervous system from damage in neurodegenerative diseases. Thus, this study sought to compare the therapeutic potential of mesenchymal cells and pericytes, both obtained from the same human adipose tissue, in SOD1 mice. For this purpose, survival and physical performance (weight, PaGE, motor score and rotarod) were evaluated. Except for the benefits observed in PaGE and the motor score tests in an early stage of the disease, treatment with MSCs and pericytes does not result in significant effects on disease progression of SOD1 female mice. For males, treatment with pericytes stands out compared to treatment with MSCs or HBSS (vehicle), resulting in beneficial effects on survival and in certain physical functions of the animals, particularly for the motor score and rotarod tests, where improvement was observed in the initial stage of the disease. The analysis of gene expression in the brain and spinal cord in end-stage animals suggests that treatment of males with pericytes can stimulate the animals\' antioxidant defense. No traces of injected human cells were observed in brain or spinal cord of mice, indicating a possible systemic effect of the transplant

A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa de caráter progressivo caracterizada pela morte seletiva de neurônios motores que leva rapidamente os pacientes à morte. O camundongo transgênico que expressa a superóxido dismutase 1 humana mutante é o modelo experimental mais aceito para o estudo da doença. Os mecanismos que levam a perda neuronal ainda são pouco conhecidos e não existe tratamento eficaz para prolongar a vida do indivíduo. Estudos recentes indicam que as células gliais aceleram o processo neurodegenerativo, entretanto os mecanismos moleculares ainda não estão estabelecidos. Os astrócitos merecem uma atenção particular, pois apresentam íntima interação com os neurônios, fornecendo suporte estrutural, metabólico e trófico. Além disso, participam ativamente da modulação excitatória neuronal e da neurotransmissão, controlando os níveis extracelulares de íons e neurotransmissores. O presente estudo propôs investigar in vitro os possíveis dos astrócitos extraídos da medula espinal do camundongo de idade neonatal (P1) e adulta pré-sintomática (P60) sobre a morte de neurônios motores na ELA. Para isso, o trofismo e sobrevida do neurônio motor espinal foram avaliados nas culturas de neurônios motores tratados com meio condicionado de astrócitos e também em sistemas de co-culturas neurônios motores/astrócitos, de origem SOD1G93A (transgênica) e selvagem (wild type) em diferentes combinações. Investigou-se ainda, a expressão gênica de genes nos astrócitos nas culturas P1 e P60 realizadas através do PCR quantitativo (qPCR) e a quantificação de moléculas secretadas pelos astrócitos por ELISA Sanduíche. Para o estudo do trofismo e degeneração neuronal, as células foram marcadas com marcadores específicos de morte neuronal e o trofismo dos neurônios também foi quantificado por contraste de fase. As células foram quantificadas por métodos estereológicos específicos e as análises mostraram que o tratamento com meio condicionado de astrócitos transgênicos P1 e P60 causaram respectivamente retrações nos prolongamentos e morte dos neurônios transgênicos. As análises da morte neuronal dos meios condicionados e co-cultura mostraram que os astrócitos transgênicos de ambas as idades causaram a morte de neurônios wild type e apenas os astrócitos transgênicos P60 levaram os maiores perfis de morte nos neurônios transgênicos, demonstrando a toxidade dessas células. Quanto à análise da expressão gênica, os genes NKRF, UBE2I e TGFA mostraram-se diferencialmente expressos nos astrócitos transgênicos P1 e os genes HIPK3, TGFA e NTF5 diferencialmente expressos nos astrócitos transgênicos P60. Nas análises das moléculas secretadas nos meios condicionados maior quantidade de NGF foi encontrada no meio dos astrócitos transgênicos P60 comparando-se aos astrócitos wild type. A quantidade de IGF-I diminuiu no meio condicionado da cultura de astrócitos transgênicos P60 comparando-se aos astrócitos wild type E ainda, há a diminuição autócrina de TNF-? e IL-6 nos astrócitos transgênicos P60. Os astrócitos transgênicos parecem promover a toxicidade ao neurônio motor na ELA e moléculas liberadas pelos astrócitos parecem estar envolvidas no processo de desenvolvimento da ELA / Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neurons death that readly leads patients to death. Transgenic mice expressing human mutant superoxide dismutase 1 (hSOD1) is the most accepted experimental model for the disease studying. The mechanisms that lead to neuronal loss are still poorly understood and there is no effective treatment able to prolong the pacient\'s life. Recent studies indicate that glial cells accelerate the neurodegenerative process, but the molecular mechanisms are not yet established. Astrocytes deserve particular attention, since they have close interaction with neurons, providing structural, metabolic and trophic support. In addition, they also participate actively in the neuronal excitatory modulation and in neuronal transmission, controlling ions and neurotransmitters at extracellular levels. The present study aimed to investigate the possible in vitro effects in astrocytes on the motor neurons death in ALS from newborn (P1) and adult pre symptomatic (P60) spinal cord mice. Thus, we evaluated spinal motor neuron survival and trophism in cultures treated with astrocytes conditioned medium and also in co-culture neuron/astrocyte systems of SOD1G93A (transgenic) and wild type in different cells combinations. Still, we investigated genes expression related to P1 and P60 astrocytes cultures performed by quantitative PCR (qPCR) and the molecules secreted by astrocytes were quantified by Sandwich ELISA. For the neuronal degeneration and trophism study, cells were immunostained with specific markers and neurouns were also visualized by phase contrast. These cells were quantified by stereological method and their analysis showed that treatment with transgenic P1 and P60 astrocytes conditioned medium cause length retractions and death on transgenic motor neuron. But, the neuronal death on conditioned medium and co-cultures experiments showed that transgenic P1 and P60 astrocytes caused wild type neuronal death and only transgenic P60 astrocytes led transgenic neurons death, demonstrating major toxicity of transgenic astrocytes. For the gene expression analysis NKRF, UBE2I and TGFa genes showed differentially expressed in transgenic P1 astrocytes and HIPK3, TGFa and NTF5 genes showed differentially expressed in transgenic P60 astrocytes. The analizes of molecules secreted by conditioned media a larger amount of NGF was found in transgenic P60 astrocytes comparing to wild type astrocytes. The amount of IGF-I in the conditioned medium was reduced in astrocytes transgenic P60 cultures compared to the wild type astrocytes Also, there is a reduction autocrine of TNF-? and IL-6 on transgenic astrocytes P60. The transgenic astrocytes seem to promote motor neuron toxicity in ALS and molecules released by astrocytes appear to be involved in the ALS development

Bakgrund:Amyotrofisk lateralskleros (ALS) tillhör gruppen neurologiska sjukdomar. Den har ett snabbt förlopp och går inte att bota. I Sverige insjuknar 220-250 invånare varje år och 750-850 invånare beräknas ha sjukdomen idag. Medelålder är 45-75 år men kan debutera i alla åldrar. Efter sjukdomens första symtom ses en förväntad livslängd mellan två till tre år.Syfte:Att belysa patienters upplevelser av att leva med ALS. Metod:Denna litteraturstudie är av induktiv ansats med integrerad analys och baseras på 8 vetenskapliga artiklar som speglar människors upplevelse. Sökningar gjordes i databaserna CINAHL och PubMed.Resultat:I analysen identifierades två huvudkategorierAtt förlora fotfästet med underkategorierna att tappa kontrollen ochatt hamna i beroendeställning.ALS innebär en stor förändring i livet med blandade känslor som att inte kunna kontrollera sin egen kropp. Att möta problemen med underkategoriernaatt accepteraoch att möta någon med samma diagnos. Det är viktigt att försöka acceptera situationen och göra det bästa av tiden som finns kvar. Slutsats:ALS innebär både positiva och negativa upplevelser. Livet är inte längre en självklarhet och tiden blir dyrbar. Det är viktigt att som sjuksköterska ha bra kunskap om sjukdomen och vårda med livsvärlden som fokus.

A Esclerose Lateral Amiotrófica é uma doença neurodegenerativa que afeta seletivamente neurônios motores. A maior parte dos casos de ELA (90%) é esporádica. Para os casos familiais, mais de vinte genes já foram associados. Diversos mecanismos estão envolvidos na patogênese, entre eles o estresse oxidativo, proteostase e agregação, excitoxicidade, tráfego intracelular, entre outros. A mutação P56S na proteína VAPB está associada à ELA8. A VAPB é uma proteína de membrana do retículo endoplasmático e está possivelmente envolvida em diversas funções celulares, dentre elas tráfego intracelular, interação retículo endoplasmático-aparelho de Golgi e UPR. Sabendo que mutações no gene que codifica VAPB resultam em ELA e que indivíduos com a mesma mutação neste gene podem apresentar quadros clínicos bastante diferentes, propõe-se estudar a suscetibilidade ao estresse oxidativo e ao estresse do retículo endoplasmático e as possíveis vias de degradação das proteínas mutantes como fatores subjacentes a essa heterogeneidade clínica. Desta forma, objetivou-se realizar uma análise integrada de ELA8, buscando compreender os mecanismos moleculares envolvidos na doença, utilizando a levedura Saccharomyces cerevisiae como modelo para estudo. Foram obtidas diferentes linhagens BY4741 de S. cerevisiae, expressando os genes de VAPBWT e VAPBP56S ou o plasmídeo vazio (que foi utilizado como controle em todos os experimentos) sob controle do promotor GAL1. Foram avaliadas as viabilidades das células expressando as proteínas humanas, a sua localização celular e possível formação de agregados. Os resultados mostram que a expressão da proteína VAPBP56S é tóxica e leva à formação de agregados dispersos nas células, enquanto a expressão da proteína selvagem se concentra no retículo endoplasmático e não altera significativamente a viabilidade das células. Scs2 é a proteína de levedura homóloga a VAPB, e a deleção do gene correspondente gera linhagens auxotróficas para inositol. VAPBWT e VAPBP56S foram expressas em linhagem nocaute para o gene Scs2, a fim de analisar se os homólogos humanos complementam a auxotrofia a inositol. Observou-se que a linhagem expressando a proteína selvagem é capaz de restaurar o fenótipo selvagem e a linhagem expressando a proteína mutante não. Para avaliar os efeitos de estresse oxidativo nas linhagens BY4741, foram determinados: a viabilidade e sensibilidade das linhagens sob condições de estresse induzido por H2O2, a razão GSH/GSSG e a produção de H2O2 por mitocôndrias, além da viabilidade após tratamento com o antioxidante N-acetil-L-cisteína. De modo geral foi verificado que a linhagem expressando a proteína mutante é discretamente mais sensível ao tratamento com H2O2, possui menor razão GSH/GSSG, e produz mais H2O2 em mitocôndrias isoladas. Em conjunto, estes dados sugerem alteração no metabolismo redox a partir da expressão de VAPBP56S. Os efeitos da inibição do proteassomo (ΔPdr5 + MG132) e da autofagia (ΔAtg8) foram avaliados em ensaios de viabilidade e degradação proteica. Verificou-se que a inibição do proteassomo tem maior efeito sobre a viabilidade das linhagens expressando VAPBWT e diminui a degradação desta proteína. A inibição da autofagia, ao contrário, afeta mais a linhagem expressando VAPBP56S. A atividade do proteassomo, a ubiquitinação de proteínas e os níveis de autofagia também foram avaliados, sendo verificado que há maior expressão de subunidades do proteassomo nas linhagens expressando ambas as proteínas. Na linhagem expressando VAPBWT, observou-se maior atividade do proteassomo e uma diminuição no pool de proteínas ubiquitinadas, de acordo com a maior expressão de subunidades do proteassomo. Na linhagem expressando VAPBP56S, ao contrário, há diminuição da atividade do proteassomo e acúmulo de proteínas ubiquitinadas, sugerindo uma inibição do proteassomo. Por meio do monitoramento da fusão GFP-Atg8 foi verificada a maior formação de autofagossomos nas linhagens expressando VAPBP56S, o que sugere maiores níveis de autofagia. Foi avaliada a viabilidade das células sob o efeito do aumento da expressão de Tsa1, uma peroxirredoxina com capacidade de recrutar chaperonas para agregados de forma redox dependente. Observou-se que esta proteína é capaz de atenuar a toxicidade de VAPBP56S, especialmente no ensaio de diluição seriada. Por fim foram verificados os níveis de marcadores de estresse do retículo endoplasmático, Pdi1, Ero1, Lhs1 e Kar2, e de UPR, SHac1, e foi visto que a expressão da proteína mutante alterou todos estes indicadores. Os dados em conjunto sugerem alterações no metabolismo redox e na proteostase resultantes da expressão de VAPBP56S / Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects motor neurons. The majority of ALS cases (90%) are sporadic. More than twenty genes have been associated with familial cases. Several mechanisms are involved in ALS pathogenesis, including oxidative stress, proteostasis and aggregation, excitotoxicity, intracellular trafficking, and others. The P56S mutation in the protein VAPB is associated with ALS8. VAPB is a membrane protein of the endoplasmic reticulum that is possibly involved in diverse cellular functions, including intracellular trafficking, interaction endoplasmic reticulum-Golgi and UPR. Knowing that mutations in the gene encoding VAPB result in ALS and that individuals with the same mutation in this gene can show different clinical conditions, we aimed to analyze the susceptibility to oxidative and endoplasmic reticulum stresses and protein degradation pathways as factors underlying this clinical heterogeneity. Thus, the objective was to perform an integrated analysis of ALS8, trying to understand the molecular mechanisms involved in the disease using, for this purpose, budding yeast Saccharomyces cerevisiae was employed as a model for this study. Different strains of S. cerevisiae containing the gene VAPBWT or VAPBP56S or the empty plasmid were obtained. BY4741 strains were evaluated for their viability when expressing human proteins, the subcellular localization of these proteins and the ability to form aggregates. The results show that the expression of the mutant protein is toxic and leads to the formation of disperse aggregates in the cell, while the expression of the wild-type protein is concentrated in the endoplasmic reticulum and does not alter cell viability. Scs2 is the yeast homologue of VAPB and deletion of the corresponding gene renders cells auxotrophic for inositol. Therefore, VAPBWT and VAPBP56S genes were expressed in Δscs2 cells to evaluate their ability to complement the inositol auxotrophy. The strain expressing the wild-type protein was able to restore the wild type phenotype, while the strain expressing the mutant protein was not. To evaluate oxidative stress in BY4741 strains expressing human proteins, it was determined: viabilities and sensitivities to stress induced by H2O2; GSH/GSSG ratio and H2O2 production in the mitochondria, as well as viabilities after treatment of cells with N-acetyl-L-cysteine. In general, it was found that the strain expressing the mutant protein is slightly sensitive to treatment with H2O2, had minor GSH/GSSG ratio, which indicates more oxidative cellular environment, and has a major production of H2O2 in isolated mitochondria. Together, these data suggest important changes in the redox metabolism associated with VAPBP56Sexpression. The effects of inhibition of the proteasome (ΔPdr5 + MG132) and autophagy (ΔAtg8) were evaluated through viability assays and protein degradation. Inhibition of the proteasome had a greater effect on the viability of strains expressing VAPBWT and decreased the degradation of this protein. Inhibition of autophagy, in contrast, mainly affected the strain expressing VAPBP56S. The activity of the proteasome, protein ubiquitilation and autophagy levels were evaluated in BY4741 strains expressing human proteins. We found an increased expression of proteasome subunits in the strains expressing both proteins, which lead to an increased activity of proteasome in VAPBWT strain and a decrease in the pool of ubiquitilated proteins. In strain expressing VAPBP56S instead, there is a reduced proteasome activity and accumulation of ubiquitilated proteins. By monitoring the GFP-Atg8 fusion, it was verified that the formation of autophagosomes was increased in strains expressing VAPBP56S, suggesting higher levels of autophagy. The effect of Tsa1 expression, a peroxiredoxin capable to recruit chaperone to aggregates, on cell viability was evaluated and it was observed that this protein was able to attenuate the toxicity of VAPBP56S. Finally the levels of endoplasmic reticulum stress markers, Pdi1, Ero1, Lhs1 and Kar2, and the UPR marker, SHac1, were checked and it was found that the expression of the mutant protein is able to change all these indicators. Taken together, our data suggest changes in the redox metabolism and proteostasis linked to VAPBP56S expression

A Esclerose Lateral Amiotrófica (ELA) é uma doença progressiva caracterizada pela perda de neurônios motores levando rapidamente os pacientes à morte. Os mecanismos da morte neuronal são desconhecidos e estudos recentes indicam que a microglia pode participar deste processo. Para investigar o papel da microglia na ELA, camundongos transgênicos SOD1G93A foram utilizados na avaliação da morte e do trofismo neuronal em sistemas de co-culturas neurônio/microglia e do efeito do meio condicionado microglial sob os neurônios motores. Ambas as células foram extraídas da medula espinal de camundongos transgênicos (TG) e wild type (WT). A microglia foi obtida dos animais neonatos e adultos na fase pré-clínica da doença. Os neurônios extraídos de neonatos foram marcados com reagente específico para morte neuronal e seus prolongamentos foram quantificados em contraste de fase por métodos estereológicos específicos. A expressão gênica de moléculas candidatas à participação do processo neurodegenerativo relacionadas com a microglia foi quantificada pelo PCR em tempo real, assim como a quantidade de moléculas secretadas no meio condicionado das culturas microgliais dosada pelo ELISA sanduíche. O meio condicionado da microglia TG neonatal não foi capaz de acentuar a morte neuronal, entretanto a neurodegeneração foi potenciada nas análises das co-culturas. Interessantemente, o meio condicionado das microglias WT provenientes de animais adultos foram capazes de aumentar os prolongamentos neuronais feito não observado em relação às microglias TG. Quantidades aumentadas do fator de necrose tumoral ?, interleucina-6 e fator de crescimento derivado do nervo foram quantificadas no meio condicionado das microglias TG. A microglia TG neonatal apresentou dimuição na expressão gênica de AKAP13, HIPK3, UBE2I E NTF5. Esses achados precoces sugerem perda de migração microglial, perda de resistência à apoptose, desbalanço entre proliferação e morte celular e diminuição do suporte trófico neuronal. Em conjunto, os resultados evidenciam a participação da microglia nos mecanismos da ELA / Amyotrophic Lateral Sclerosis (ALS) is a progressive degenerative disorder affecting motoneurons and leading the patient to death.The cause of motor neurons degeneration in ALS is uncertain and there is any treatment able to prolong the patient life. Recent studies show that microglia could participate of the process of ALS degeneration. Its activation is linked to the disbalance of neuroprotective and neurotoxic action. To investigate the microglia role in ALS, SOD1G93A transgenic mice that develop symptoms similar to the clinical disease were used. We evaluated the neuronal death and trophism in microglia/neuron co-cultures system and in microglial conditioned medium effect under the neurons. Neurons and microglia were extracted from transgenicor wild type mice spinal cord. Microglia was obtained from 1 day pos natal pups and adult onset of disease mice. Cells were stained with a specific marker to neuronal death. Neuronal extensions area and neuronal death was quantified by stereological method. The genic expression of candidate molecules related to degeneration in ALS was quantified by real time PCR and the release of some molecules were quantified by ELISA sandwich. Results showed that maybe transgenic neonatal microglia is not able to increase the neuronal death through releasing molecules in its conditionated medium, on the other hand when transgenic microglia was co-cultured with any kind of neuron, neuronal death was observed. Microglia from adult transgenic mice was not able to promote a neuroprotection compared to wild type in co-culture and conditionated medium experiments. In addition to this, was observed increased tumor necrosis factor alpha, interleukin 6 and nerve growth factor secretion by transgenic microglia. Neonatal transgenic microglia also exhibited reduced genic expression of AKAP13, HIPK3, UBE2I and NTF5. These findings at an early stage suggest a lost of migration potential, lost of apoptosis resistance, disbalance of proliferation and cell death and reduction of neuronal trophic support. So together, these data indicate the involvement of microglia in ALS mechanisms