Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation

Drosophila models of neuromuscular disorders: From morphology to behavior

RESUMO: As doenças neurodegenerativas, que podem originar ataxia ou demência, são doenças incuráveis e debilitantes que têm como consequência a degeneração progressiva seguida de morte neuronal. O mecanismo que provoca a neurodegeneração não é totalmente conhecido, sabendo-se apenas que está associado a múltiplas vias e contribuições genéticas. Dada a complexidade e variedade de mecanismos patológicos, um tratamento eficaz para este tipo de distúrbio é difícil de alcançar. Uma forma de contornar esta dificuldade, é através da promoção da complexidade neuronal de células ainda funcionais como meio de retardamento da progressão da doença. Ao caracterizar a morfologia e composição sináptica de vários modelos de neurodegeneração usando Drosophila melanogaster, o objetivo é averiguar se existe um alvo específico comum que possa retardar este sintoma. Para isto, foi utilizado como recurso um knocdown neuronal de genes associados a duas doenças neuromotoras, a Neurodegeneração associada à cinase do Ácido Pantoténico e a Paraplegias Espáticas Hereditárias. Para além disto, análises comportamentais locomotoras utilizando vários ensaios com diferentes sensibilidades foram realizadas em várias fases do ciclo de vida da mosca da fruta, através da utilização de knocdown específico para neurónio e células da glia para modelos de Esclerose Lateral Amiotrófica, Atrofia Muscular Espinhal, PKAN e HSP. Aqui, verificou-se que todos os genótipos estão associados a uma função locomotora diminuída que parece ser dependente das proteínas que foram anuladas nos neurônios e na glia. Este trabalho contribui para a compreensão da composição sináptica dos modelos de doença acima referidos que poderá ser utilizado como fator determinante para encontrar uma via que contorne a neurodegeneração através do aumento da complexidade neuronal. Para a PKAN, dado que muito pouco se sabe acerca desta doença, este trabalho contribui para a perceção de mais uma via que induz neurodegeneração e quais as alterações sinápticas possivelmente associadas.ABSTRACT: Neurodegenerative disorders are one of the most common illness manifestations worldwide. They have a chronic and progressive display, characterized by synaptic malfunction that then evolve to irreversible neuronal death. The mechanism behind neurodegeneration is not fully understood and can have several associated pathways and genetic contributions. Given that a treatment suitable for all forms of neurodegeneration is hard to get, one idea to overcome this issue is to promote neuronal complexity in the non-affected neurons, to try to delay the progression of these type of disorders. By characterizing the synaptic composition of several examples of Drosophila melanogaster models of neurodegenerative diseases, we intended to explore whether there is a specific target that can be targeted to slow neurodegeneration. Using cell type specific RNAi, we induce neuronal knockdown of genes associated with Pantothenate Kinase Associated Neurodegeneration and Hereditary Spastic Paraplegia. Several features of synaptic morphological and composition were addressed at the Drosophila NMJ. Also, behavior analyses using several assays with different sensitivities were performed in several stages of the Drosophila life cycle. Using neuronal or glial specific RNAi against proteins associated with Amyotrophic Lateral Sclerosis, Spinal Muscular Atrophy, PKAN and HSP, we show that all of these genotypes are associated with impaired locomotor function that seems to be dependent on the proteins that were knockdown in neurons and in wrapping glia cells. This work contributes to the understanding of the synaptic composition of HSP and PKAN disease models, that can be used to help us target downstream pathways, that will lead to increase structural plasticity as a way to promote neuronal complexity and overcome neurodegeneration. For PKAN, as very little information is available, it contributes to the understanding of the underlying causes of the disorder and to provide insights in which are the synaptic alterations behind patients with mutations in PanK2

UWOMJ Volume 69, No 2, Spring 1999

An interdisciplinary medical science publication; established 1930.https://ir.lib.uwo.ca/uwomj/1030/thumbnail.jp

Wisp1 is associated with hepatitis B related human hepatocellular carcinoma and promotes proliferation or migration of HCC derived cell lines

Worldwide, primary hepatocellular carcinoma is the fifth most commonly diagnosed solid cancer and the third most common cause of cancer related death. Because of its constantly increasing incidence in developed countries and its poor prognosis, HCC represents a major health problem. Diagnostic markers for early-stage HCC are of great need but still lacking due to the high complexity and heterogeneity of HCC. The purpose of this study was to examine Wisp1 expression in different liver tumor stages and its functional role in epithelial- and fibroblastoid-type HCC cell lines. By immunohistochemical staining of HCC patient samples, we demonstrated that Wisp1 expression is enhanced in well- and moderately differentiated HCC and is associated with inflammation and steatosis, but is absent in poorly differentiated HCC. Furthermore, we found that Wisp1 is expressed and secreted in HCC cell lines(FLC4, HLF). Treatment of the epithelial-type HCC cell lines with human recombinant Wisp1 led to an increase in proliferation, while fibroblastoid-type HCC cell lines showed an increase in migration. Interestingly, both types of cell lines displayed the activation of the same phosphokinases upon Wisp1 stimulation. Knockdown of Wisp1 with shRNA in the epithelial-type HCC cell line consequently decreased proliferation and, more importantly, led to apoptosis. Our study thus identified Wisp1 as a novel immunohistochemical marker for the detection of well differentiated, early-stage HCC as well as a regulator of proliferation in the epithelial-type HCC cell line and a regulator of migration in the fibroblastoidtype HCC cell lines via activation of distinct HCC related phosphokinases. These results pose Wisp1 as a possible diagnostic marker for HCC for which currently only poor therapies are available

Study of mechanisms of methylmercury neurotoxicity and association with Parkinson's disease

Methylmercury (MeHg) is a well-known neurotoxicant but the diverse mechanistic events associated with MeHg neurotoxicity have yet been fully elucidated. In this study, we explored the mechanisms of MeHg neurotoxicity particularly its possible roles in neurodegenerative diseases like Parkinson’s disease (PD) using dopaminergic neuronal cells and a non-human primate model. In the cell culture model, we compared effects of MeHg to those induced by 1-methyl-4-phenylpyridinium (MPP⁺), a well-established drug that can induce Parkinsonism-like symptoms. A proteomic approach was used to identify and analyze MeHg affected proteins and their biological functions and associated pathways in both the cellular and marmoset models. Our results showed that MeHg induced changes of gene/protein profiles are similar to the effects as MPP⁺. Evidence from proteomic results suggested MeHg caused neurodegenerative effects not only associated with PD but also other neurodegenerative disorders such as Huntington’s disease (HD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS). We also found brain regional specific response to MeHg stimuli, based on the protein profiles affected in the following order: cerebellum > occipital lobe (OL) > frontal lobe (FL) of the cerebrum. In the cerebellum, carbohydrate derivative metabolic process, synaptic transmission, cell development and calcium signalling are dominant functions and pathways contributing to the motor deficit in MeHg-treated marmoset. MeHg was found to selectively target membrane proteins in the cerebellum particularly in synaptic membranes. MeHg affected proteins involved in energy metabolism in both OL and FL of the cerebrum through different proteins and biochemical pathways. In the OL, proteins were enriched in functions of carbohydrate metabolic process, lipid metabolic process, cellular amino acid metabolic process, homeostatic process, transportation, and regulation of body fluid level. In the FL, differentially expressed proteins were mainly involved in the cell cycle and cell division, glycerolipid metabolic process, sulfur compound metabolic process, cellular amino acid metabolic process, microtubule-based process, and proteolysis. The dyshomeostasis of water transport and associated pathways observed in OL and FL was found to be the underlying mechanism for brain edema observed in the MeHg exposed marmoset. Novel proteins such as DLG4 (PSP95) in the cerebellum and APOE in OL were exhibited to be core proteins in linking multifunction targeted by MeHg. This study provides a new perspective upon understanding mechanisms behind MeHg mediated neurotoxic deficits, and suggests potential links between MeHg exposure and neurodegenerative disorders in humans.The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b207397

The protective protein : a multifunctional lysosomal enzyme

This thesis describes the characterization of a lysosomal protein, the 'protective protein', that has at least two functions. On the one hand it protects lysosomal ~galactosidase and neuraminidase from degradation within the lysosome, hence its name. On the other hand it has peptidase and deamidase activities, that could be involved in protein turnover in lysosomes and hormone (in)activation. Degradation is distinguished here from proteolytic processing, although both involve peptide hydrolysis. The first is, however, an aspecific random process, carried out at multiple sites, whereas the second is a highly specific (single) event. Given the putative function of the protective protein it seemed appropriate to start with an overview of intracellular sites of protein degradation, followed by a section on the biogenesis of lysosomes. The introduction ends with a summary on what is known about lysosomal storage disorders, a group of genetic diseases that are due to defects in lysosomal proteins. The protective protein itself is impaired in the rare disorder galactosialidosis and studies on this disease have been the basis for the discovery of the protein and analysis of its functions