Master of Science / Department of Biology / Stella Y. Lee / CLN5 is one of several proteins that when mutated result in the lysosomal storage disorder (LSD) Neuronal Ceroid Lipofuscinosis (NCL). CLN5 is a soluble lysosomal protein that has no known function at this time. Previously we showed that eight asparagine residues in CLN5 are N-glycosylated, and that this modification is important for the protein’s transport and function. Now, we have identified a link between the activation of autophagy and CLN5 deficiency. The autophagy-lysosomal protein degradation system is one of the major pathways the cell uses to degrade intracellular material and recycle cellular building blocks. It was recently shown that other CLN proteins affect the relative level of autophagy, indicating a potential link between the autophagy pathway and the NCLs. By knocking down endogenous CLN5 in HeLa we showed that, upon stress induction, cells responded with higher levels of autophagy activation. Consistent with these knockdown experiments, there is a higher level of the autophagy marker protein, LC3-II, in CLN5 patient cells that are naturally deficient for the CLN5 protein. Pharmaceutical induction of autophagy through different means also showed higher LC3-II levels compared to control, though patterns differed in the type of autophagy induced. In summary, we discovered that the autophagy pathway is altered in CLN5 deficient cells, indicating a potential role for CLN5 in autophagy. Further analyses of the autophagy pathway will shed light on where CLN5 is acting and the mechanism by which defective CLN5 causes NCL.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Stella Yu-Chien Lee / Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative lysosomal storage disorders which is the most frequent group of inherited neurodegenerative disorders that affect children leading to severe pathological conditions such as progressive loss of motor neuron functions, loss of vision, mental retardation, epilepsy, ataxia and atrophy in cerebral, cerebella cortex and retina and eventually premature death. Among the many genes that cause NCL, mutations in CLN5 leads to different forms of NCL (infantile, late infantile, juvenile and adult) and mutations in CLN8 leads to progressive epilepsy with mental retardation (EPMR) and a variant late infantile form of NCL. The function(s) of both CLN5 and CLN8 proteins remain elusive. CLN5 is a glycosylated soluble protein that resides in the lysosome. We observed that endogenous CLN5 protein exist in two forms and identified a previously unknown C-terminal proteolytic processing event of CLN5. Using a cycloheximide chase experiment we demonstrated that the proteolytic processing of CLN5 is a post-translational modification. Furthermore treatment with chloroquine showed the processing occurs in low pH cellular compartments. After treatment with different protease inhibitors our results suggested the protease involved in the processing of CLN5 could be a cysteine protease. Using two glycosylation mutants of CLN5, retained in the endoplasmic reticulum (ER) or the Golgi we showed the proteolytic processing occurs in an organelle beyond the ER. This study contributes to understanding the characteristics of the CLN5 protein. CLN8 is an ER resident transmembrane protein that shuttles between the ER and the ER-Golgi intermediate compartment (ERGIC). In our study we identified a potential interaction between CLN8 and a PP2A holoenzyme complex consisting regulatory subunit A α isoform and regulatory subunit B α isoform. Using two CLN8 patient derived fibroblast cell lines we were able to show that the phosphorylated levels of PP2A target kinase Akt was reduced at both of its regulatory sites Ser473 and Thr308 and the activity of PP2A was increased. A delay of ceramide transport from ER to Golgi in CLN8 deficient patient cell lines was observed using BODIPY FL C5-Ceramide staining. Our results provide evidence for CLN8 protein being involved in the regulation of PP2A activity and trafficking of ceramide from ER to Golgi.

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A estrutura plástica do cérebro tem a capacidade de se adaptar a diversas condições e estímulos. No entanto, isso também pode facilitar a emergência de instabilidades, o que acarreta na necessidade de mecanismos de homeostase que previnam que a dinâmica da rede neural chegue a estados patológicos. A plasticidade associativa é considerada a principal base para o desenvolvimento de funções como memória e aprendizado, a realimentação positiva potencialmente leva à saturação de sinapses e instabilidades de atividade, especialmente em arquiteturas om conectividades recorrentes tais como em microcircuitos cerebrais. Neste trabalho investigamos a difícil interação entre a codificação de informação e o controle da atividade através da plasticidade Hebbiana e do escalonamento sináptico homeostático. O objetivo é a determinação de propriedades, como por exemplo a inibição e a conectividade, que proporcionam o desenvolvimento de codificação de informação de uma maneira confiável e fisiologicamente relevante através de plasticidade sináptica, prevenindo comportamento patológico. Após uma breve revisão bibliográfica de tópicos básicos da neurofisiologia e da modelagem de redes neurais, a primeira parte dos resultados apresenta uma rede que, sob uma forma específica de esc alonamento sináptico, desenvolve associatividade de padrões de disparo espaço-temporais e discute a afetação da capacidade de separação e confiabilidade de acordo om escalas de tempo de plasticidade, limitações sobre a eficácia sináptica e a dinâmica das interações inibitórias. A segunda parte define condições para manter o escalonamento sináptico homeostático sem instabilidades dinâmicas, om foco em fenômenos pouco explorados, como o escalonamento de sinapses inibitórias e o alcance efetivo da plasticidade. Em direção a outros mecanismos que podem influenciar esse balanço, a última parte descreve os efeitos do local de expressão da plasticidade de longa duração sobre a dinâmica de aprendizado, o que é demonstrado diferir de acordo om a codificação do estímulo.

O protocolo conhecido como Dynamic Clamp consiste em utilizar um computador para introduzir condutâncias artificiais em um neurônio biológico. O modo como estas condutâncias dependem da voltagem da membrana ou do tempo são modelado por equações diferenciais que são integradas em tempo real por um computador conectado ao neurônio biológico. Resumidamente, o computador tem acesso ao potencial de membrana dos neurônios através de eletrodos intracelulares conectados a conversores analógico-digitais (ADCs), calcula as correntes a serem injetadas nos neurônios e produz os sinais de saída através de conversores digitalanalógicos (DACs) que produzem a injeção das correntes nos eletrodos intracelulares. De um certo modo, o Dynamic Clamp utiliza os neurônios como simuladores, permitindo investigar a importância de um tipo de condutância para a atividade elétrica de um neurônio, assim como determinar o efeito produzido pelas sinapses em uma rede, combinando o controle e flexibilidade de uma simulação no computador com a acurácia e o realismo de um experimento em eletrofisiologia. Descrevemos a implementação de um protocolo de Dynamic Clamp utilizando um computador pessoal tipo IBM-PC que permitiu contornar 3 das principais limitações que apresentam alguns dos programas de Dynamic Clamp comerciais/gratuitos disponíveis atualmente: (a) Garantia de que o sistema roda em tempo real - nossa implementação é baseada em um programa de Dynamic Clamp que roda em uma plataforma Linux Real-Time que além de controlar os experimentos em tempo real consiste em software livre com codigo fonte aberto e que pode ser instalado gratuitamente; (b) Não necessita de hardware de aquisição de dados dedicado para eletrofisiologia - utilizamos uma placa ADC/DAC comercial comum marca National Instruments modelo PCIMIO16E4. Com o driver COMEDI instalado para placas de aquisição de dados Linux, a maioria das placas ADC/DAC tipo PCI disponíveis no mercado podem ser utilizadas em implementações futuras; (c) Aumentar o número de neurônios que podem ser conectados simultaneamente - desenvolvemos um circuito demultiplex analógico que permite controlar até 8 neurônios biológicos/artificiais a partir das duas saídas analógicas que as placas DAC comerciais possuem e ainda atingir frequências de atualização da corrente de até 3 kHz (para 8 correntes de saída). Apresentamos os resultados de diversos testes que fizemos usando o programa adaptado e o circuito demultiplex para produzir sinapses em tempo real e conectar diversos neurônios artificiais em pequenas redes. Também mostramos alguns resultados preliminares obtidos com a primeira implementação de um modelo de neurônio estocástico tipo Hodgkin-Huxley em um programa de Dynamic Clamp. / The Dynamic Clamp protocol consists in using a computer to introduce artificial conductances in a biological neuron. The voltage- and time-dependency of each conductance is modeled by differential equations integrated in real-time by the computer connected to the biological neurons. In short, the computer executes a 3-phase cycle in which the membrane potential of the neurons is measured by intracellular electrodes and digitized by analog-to-digital converters (ADCs), the currents are calculated based in the digitized membrane potentials and current signals are generated by digital-to-analog converters (DACs). These currents are actually injected in the neurons by other intracellular electrodes. In some extent the Dynamic Clamp uses the neurons as simulators, allowing one to investigate the role of a specific conductance in the intrinsic activity of a neuron as well as to look for the effects of a synapse in the behavior of a small network. The Dynamic Clamp combines the control and flexibility of a computer simulation with the reality of an experiment in electrophysiology. We describe an implementation of a Dynamic Clamp protocol that allowed us to surmount 3 of the main drawbacks present in some commercial/freely available Dynamic Clamp programs: (a) Runs in real time - our implementation is based in a program that runs in a Real-Time Linux platform. This operating system not only ensures the experiments will be controlled in real time but also consists in open source software that can be freely downloaded and installed; (b) No need of special electrophysiology acquisition hardware - we used a commercial ADC/DAC acquisition board model PCI-MIO16E4 from National Instruments. With the COMEDI Linux package driver that is used most of the PCI commercial ADC/DAC boards can be used in future implementations with no change needed in the program itself. (c) We can connect more than two neurons with artificial synapses - we developed an analog demultiplex circuit that allowed us to control simultaneously up to 8 biological/artificial neurons from the two analog outputs available in most of the commercial ADC/DAC boards and we could still reach current update rates of about 3 kHz (for 8 current outputs enabled). We present the results of several tests we performed using the program adapted to control the analog demultiplex to establish synapses and to connect several artificial neurons in small neural networks. Preliminary results from the first implementation of a stochastic whole cell Hodgkin-Huxley model neuron in a real time Dynamic Clamp program are also shown.

Submitted by Fernanda Rodrigues de Lima (fernanda.rlima@ufpe.br) on 2018-08-31T20:16:35Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Igor Tchaikovsky Mello de Oliveira.pdf: 2658205 bytes, checksum: fc8a40bfa31dd1cfee1f290fd4a98ae0 (MD5) / Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-09-12T21:24:45Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Igor Tchaikovsky Mello de Oliveira.pdf: 2658205 bytes, checksum: fc8a40bfa31dd1cfee1f290fd4a98ae0 (MD5) / Made available in DSpace on 2018-09-12T21:24:45Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Igor Tchaikovsky Mello de Oliveira.pdf: 2658205 bytes, checksum: fc8a40bfa31dd1cfee1f290fd4a98ae0 (MD5) Previous issue date: 2017-07-31 / CNPq / Os núcleos da base contêm circuitos neurais que, se manipulados corretamente, desencadeiam reconhecidos efeitos anticonvulsivantes. A desinibição do corpo estriado (ES) quanto a inibição da substância negra reticulada (SNPr), são anticonvulsivantes em modelos animais e compõem os denominados Sistemas anticonvulsivantes endógenos (SAES). Tanto o sistema nervoso central, quanto os SAEs podem ter suas respostas alteradas frente diferentes estímulos, pelo fenômeno de plasticidade. Apesar de ser bem conhecida a possibilidade de indução de plasticidade através de estimulação elétrica de alta frequênciae detecção de um potencial evocado num circuito neural, ainda não foi testado o possível efeito da estimulação elétrica de alta frequência no ES sobre a SNPr e sobre o comportamento do animal. Ratos machos adultos Long evans foram randomicamente separados em dois grupos: controle (n=4) e experimental (n=4), e, submetidos a cirurgia para implante de uma matriz de multieletrodos (16 canais) no hipocampo, um tetrodo (4 canais) no ES e um conjunto de eletrodos (8 canais) na SNPr para aquisição do registro de potencial de campo local. Um eletrodo de estímulo foi também implantando na via estriado nigral. Antes de cada protocolo os animais foram filmados em uma arena de acrílico durante 5 min para avaliação de possíveis alterações comportamentais geradas pelo estímulo. Por fim, os dois grupos receberam microinjeção intrahipocampal de pilocarpina para indução de eventos paroxísticos. A eletrofisiologia mostra que, após a aplicação de estímulo no ES, detectamosuma resposta evocada na SNPr. Quantificamos a média de quadrados adentrados (53,7±13,5 e 5,5±1,8), elevação (23,5±7,6 e 3,5±2,4), sacudidelas de corpo e cabeça (10,6±3,0 e zero) e autolimpeza (4,3±2,1 e zero) nos grupos experimental e controle, respectivamente. Os resultados até o presente momento sugerem que a aplicação do estímulo de alta frequência estácausando mudanças plásticas no circuito estriado nigral, o que modifica o padrão comportamental de motricidade nos animais. É possível detectar uma reposta evocada no circuito estriado nigral após a aplicação do estímulo. Temos como perspectiva analisar os dados eletrofisiológicos relacionados à microinjeção intrahipocampal de pilocarpina. / The basal ganglia contain neural circuits that, if manipulated correctly, trigger recognized anticonvulsant effects. Inhibition of the striatum (CS) and inhibition of the substantia nigra pars reticulate (SNPr), are anticonvulsants in animal models and make up the so-called Endogenous anticonvulsant mechanisms (EAMs).Both the central nervous system and the EAMs may have their responses altered in front of different stimuli, by the phenomenon of plasticity. Although the possibility of induction of plasticity through high-frequency electrical stimulation and detection of an evoked potential in a neuronal circuit is well known, the possible effect of the high-frequency electrical stimulation in the CS on the SNPr and on the behavior has not yet been tested in the animal. Long evans male adult rats were randomly divided into two groups: control (n = 4) and experimental (n = 4), and undergoing surgery to implant a microarray of electrodes (16 channels) in the hippocampus, a tetrode (4 channels ) in CS and a set of electrodes (8 channels) in the SNPr for acquisition of the local field potential. A stimulus electrode were implanted in the striatum nigral pathway. Before each protocol, the animals were filmed in an open fiel for 5 min to evaluate possible behavioral changes generated by the stimulus. Finally, the two groups received intrahipocampal pilocarpine microinjection to induce paroxysmal events. Electrophysiology shows that, after stimulus application in CS, we detected an evoked potential in the SNPr. We quantified the mean of in-squares (53.7 ± 13.5 and 5.5 ± 1.8), elevation (23.5 ± 7.6 and 3.5 ± 2.4), body and head shaking (10.6 ± 3.0 and zero) and self-cleaning (4.3 ± 2.1 and zero) in the experimental and control groups, respectively. The results suggest that the application of the high frequency stimulus is causing plastic changes in the striatum nigral pathway, which modifies the motor behavioral pattern in animals. It is possible to detect an evoked potential in the circuit after the application of the stimulus. We aim to analyze the electrophysiological data related to the intrahipocampal microinjection of pilocarpine.

Experimentos têm revelado que redes de neurônios, tanto in vitro como in vivo, mantêm atividade descrita por avalanches e se organizam em um estado crítico no qual essas avalanches são distribuídas de acordo com leis de potência. Mostramos no presente trabalho que um modelo de rede de elementos excitáveis com sinapses dinâ- micas é capaz de exibir criticalidade auto-organizada para ampla região do espaço de parâmetros. Nossos resultados estão de acordo com outros estudos que indicam que a depressão sináptica de curto prazo constitui mecanismo suciente para produzir criticalidade em avalanches neuronais. No entanto, segundo diversos pesquisadores, embora o ajuste de parâmetros seja grosso para que haja criticalidade no modelo, é mais preciso dizer que o sistema não apresenta criticalidade auto-organizada genu ína, mas sim quasi-criticalidade auto-organizada, como os demais modelos não conservativos presentes na literatura. / Experiments have shown that neuronal networks, both in vitro and in vivo, maintain activity described by avalanches and they are organized into a critical state in which these avalanches are distributed according to power laws. We have demonstrated that a model based on a network of excitable elements with dynamical synapses is able to exhibit self-organized criticality for a wide range of the parameter\'s space. Our results are consistent with other studies that suggest short-term synaptic depression is enough to produce criticality in neuronal avalanches. However, according to several researchers, in spite of the tuning to be gross to ensure that there is criticality in the model, it is more accurate do not say that the system presents genuine self-organized criticality, but self-organized quasi-criticality as the other non-conservative models in the literature.

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disorder resulting in the loss of motor neurons from the spinal cord and frontal cortex. The patterns of neurodegeneration, affected regions, age of onset, and time course of disease progression are all highly variable between and within variants of the disease. Familial ALS (fALS), inherited versions of ALS due to genetic changes, accounts for between 5-20% of all ALS cases, while the rest are sporadic, with either no causative mutation identified or no familial history of ALS. Recently, the discovery of C9orf72 hexanucleotide repeat expansions have been identified as one of the most common causes of familial ALS, with some patients presenting with dual phenotypes of ALS and frontotemporal dementia, leading to new hypotheses about the nature of neurodegenerative diseases. Despite the continued discovery of new ALS causative genes, little is known about the pathogenesis of the disease. While almost all variants include the presence of intracellular protein inclusions, the site of the protein plaques and involved proteins varies between genetic and phenotypic variants of this disease. Due to the lack of clear pathogenic mechanisms, several hypotheses have been developed to explain the process of neurodegeneration. Autophagy, the process of self-eating, leading to destruction of damaged or excess proteins and organelles, has been implicated as being altered in ALS. Multiple variants have demonstrated altered mitochondrial morphology and cellular energetic dynamics, which could explain previous observations that implicate the process of apoptosis in cellular death. Many of the involved proteins in ALS have functional roles for intracellular, nucleocytoplasmic, and axonal transport of various proteins or RNA. These three competing hypotheses are currently the most prominent hypotheses in the pathogenesis of ALS, and have largely been considered as separate and competing in past research. Is there a chance that the true pathogenesis leading to neuronal destruction via apoptosis involve all three hypotheses? Altered protein and RNA transport dynamics could lead to changes in cellular stress responses or overload autophagy pathways, leading to exacerbated cellular stress responses, leading to alterations in mitochondrial morphology and eventually cell death via apoptosis.

Cancer and neurodegeneration are detrimental conditions associated with an inappropriate regulation of cell survival and cell death, causing compromised cells to evade death or excessive death of healthy neurons. The mammalian target of rapamycin complex 2 (mTORC2) has been implicated in the regulation of cell survival by phosphorylating the protein kinase Akt. This is dependent upon the scaffold protein Sin1, a core component of mTORC2. The requirement of Sin1 in cell survival, and in particular in neuronal survival, has not been established due to the early embryonic lethality of mice with a targeted deletion of the Sin1 gene. To circumvent this issue, a novel conditional mouse knockout model was established. The role of Sin1 in regulating cell survival was evaluated in fibroblasts and cortical neurons. The loss of Sin1 significantly affected the phosphorylation and activity of Akt in fibroblasts and caused a reduction in cell survival by potentially inducing premature senescence. In contrast, the loss of Sin1 caused an increase in caspase-independent cell death in cortical neurons. Gene-expression analysis of Sin1 knockout cortical neurons demonstrated an important down-regulation of transcription factors, cytoskeletal proteins and components of signalling pathways involved in neuronal survival, aiding to uncover the mechanism by which Sin1 promotes neuronal survival. Taken together, the results presented in this study show a key role of the scaffold protein Sin1 in regulating neuronal survival.

Animal survival depends on the brain’s ability to detect the energetic state of the body and to alter behaviour in order to maintain homeostasis. Current research in the control of food consumption stresses the importance of identifying and establishing the specific roles of homeostatic neurons, which sense the body’s energetic state and elicit complex and flexible food seeking behaviours. Recent developments in optogenetics, molecular genetics, and anatomical techniques have made these investigations possible at the resolution of specific cell types and circuits. These neurons are of particular interest because they serve as key entry points to the identification of downstream circuits and reinforcement mechanisms that control feeding behaviour. This dissertation probes the role of two kinds of homeostatic neurons— agouti-related peptide (AGRP) in the arcuate nucleus (ARC) and leptin receptor (LepRb) neurons in the lateral hypothalamic area (LHA)—in the control of food intake. First, I examined the role of LepRb neurons in the LHA in feeding. Results from electrophysiological studies indicate that these neurons consist of a subpopulation of homeostatic sensing LHA γ-aminobutyric acid (GABA) expressing neurons. In addition to their response to leptin, these neurons are capable of modulating their activity in response to changes in glucose levels, further substantiating their role as homeostatic sensing neurons. Behavioural studies using optogenetic activation of these neurons show that their elevated activity is capable of reducing body weight, although their role in modulating feeding remains unclear. Second, I investigated the reinforcement mechanisms employed by AGRP neurons to elicit voracious food consumption and increased willingness to work for food. Conditioned place avoidance studies under optogenetic activation of AGRP neurons reveal that their increased activity has negative valence and is avoided. In addition, imposition of elevated AGRP neuron activity in an operant task reduced instrumental food seeking with particular sensitivity under high effort requirements. Taken together, these results suggest that AGRP neurons employ a negative reinforcement teaching signal to direct action selection during food seeking and consumption. Third, I systematically analyzed the contribution of specific AGRP neuron projection subpopulations in AGRP neuron mediated evoked-feeding behaviour. Optogenetic activation studies of AGRP neuron axons in downstream projection regions indicate that several, but not all, subpopulations are capable of independently evoke food consumption. This work reveals a parallel and redundant functional circuit organization for AGRP neurons in the control of food intake. Interestingly, all AGRP neuron subpopulations examined displayed similar modulation by states of energy deficit and signals of starvation, despite their apparent divergence in function. As a whole, this dissertation extends our understanding of the role of homeostatic neurons in food consumption and uncovers previously unappreciated functional organization and reinforcement mechanisms employed by neuronal circuits that control feeding behaviour.