The Wnt signaling network has critical roles in embryonic development and is implicated in human disease. One of the outputs of the Wnt network, called the planar cell polarity (PCP) pathway, regulates tissue polarity and directs cell migration. Core PCP components (Frizzled, Dishevelled, Prickle, Vangl, Celsr) localize asymmetrically in polarized cells and establish polarity across the tissue through protein interactions between adjacent cells. The core PCP component activate tissue-specific "effectors" which translate the signal into morphological changes. PCP is related to several disease conditions, including neural tube defects, cystic kidney disease, and cance metastasis. However, mechanisms of the PCP underlying physiological and disease-related conditions are not well understood. Here, I explore functions of the core PCP component Pk, and its relationship to disease, in the zebrafish model system. Mutations in Pk1 and Pk2 have been identified in human progressive myoclonic epilepsy patients. Pk coodinate cell movement, neuronal migration and axonal outgrowth during embryonic development. Yet, how dysfunctions of pk relates to epilepsy is unknown. Here, I show that knockdown of pk1a sensitizes the zebrafish larva to convulsant drug. To model the defects in central nervous system, I examine neurogenesis in the retina and find that both pk1a and pk2 are required for proper dendritic outgrowth in the retinal inner plexiform layer. Furthermore, I characterize the epilepsy-related mutant forms of Pk1a and Pk2. The mutant Pk1a forms show reduced ability to suppress the retinal neurogenesis defects compared to the wild-type, as well as differential ubiquitination levels. Pk2 mutant forms also show differential activities in overexpression assays and seemingly more stable proteins relative to the wild-type. Taken together, pk1a and pk2 may contribute to epilepsy by affecting neuronal patterning and thus signal processing. Another aspect of PCP function has been implicated in cilia and cilia-related disorders, also called ciliopathy. PCP effectors have been shown to modulate ciliogenesis and core PCP proteins (Vang and Dvl) regulate cilia orientation. On the other hand, cilia are not required for PCP signaling, especially asymmetric core PCP protein localization. These findings leave open the question what is the precise relationship between PCP and cilia. The Bardet Biedl Syndrome (BBS) is a type of ciliopathy that leads to obesity, retinitis pigmentosa, polydactyly, mental retardation and other symptons. A subset of BBS genes share similar knockdown phenotype in cell migration as seen in PCP knockdown embryos. Shared pehnotypes have led some to proposethat PCP and bbs genes may interact. Yet a direct relationship has yet to be established. I examine the interaction between pk2 and a central Bbs gene, bbs7. By analyzing shared phenotypes in double knockdown embryos, I find no synergistic interaction between the two, suggesting they act in distinct pathways. Bbs regulate ciliary trafficking and in zebrafish, knockdown of bbs genes leads to delayed retrograde melanosome transport. Interestingly, I find knockdown of pk2 suppresses this retrograde transport delay. Additionally, pk2 knockdown embryos show a delay in anterograde melanosome transport. These findings highlight a new role for pk2 in intracellular transport and clarifies the relationship between PCP and BBS. In summary, my work here strengthens the link between pk mutations and human epilepsy and identifies functions of pk in retinal neurogenesis and in intracellular transport. To what extent the role of neurogenesis and intracellular transport are related is worth future study. Yet, this new information provides insights into potential mechanisms of epilepsy and the relationship between PCP and BBS.

Proper control of movements is critical for an animal’s survival, and requires the robust function of a number of genetic, molecular, neuronal and biomechanical processes. This dissertation describes a body of inter-related studies utilizing a diverse collection of Drosophila mutants to probe the roles individual genes play in shaping motor pattern generation. A particular emphasis is placed on describing the consequences of genetic perturbations of voltage-gated sodium, calcium and potassium ion channels (NaV, CaV, and KV respectively) on the function of neuronal circuits that drive motor behavior. Here, I describe the development of several quantitative protocols to study alterations in of walking (IowaFLI Tracker) and flight motor program activity and behavior in Drosophila mutants. These approaches were utilized to analyze the highly-stereotypic aberrant motor program associated with electroconvulsive stimulation (ECS)-induced seizure discharge activity in each hyperexcitable mutant. Several quantitative and mechanistic similarities between flight motor program activity and ECS-evoked discharges were identified, and the distinct aberrant ECS-evoked activity disclosed an electrophysiological signature of each mutation. Ion channel mutants display a diverse spectrum of neuronal excitability phenotypes that was highlighted in a novel hyperexcitable mutant, Shaker wings down (Swd), characterized by ether-induced leg shaking reminiscent of certain KV channel mutants (e.g. Shaker, KV1) is presented. Detailed analyses revealed disrupted walking and flight, correlated with neuronal hyperexcitability and aberrant action potential generation. Surprisingly, the Swd mutation site was mapped to a single amino acid in the voltage sensor region in paralytic (para, encoding the only NaV gene in Drosophila). Genetic analysis of intra-genic heteroallelic interactions amongst Swd and other identified para alleles further revealed a number of complex mechanisms underlying a wide phenotypic spectrum of altered neuronal excitability and motor pattern generation. The effects of perturbed ion channel function on motor program generation are compared with progressive alterations associated normal aging as well as neurodegeneration. A number of age-resilient and age-vulnerable circuits were identified along with circuit-function biomarkers of aging. Throughout this study, an integrative framework utilizing non-linear dimensional reduction approaches unraveled a broader perspective to visualize and quantify similarities and distinctions between discharge phenotypes across a large collection of Drosophila mutants.

Substance abuse and mental health disorders are a leading source of years lost to disability from medical causes worldwide. Unfortunately, for most neurological disorders it is unclear how underlying genetic predispositions govern behavioral response to environmental stressors. Owing to their convenience, genetic tractability, and small brains, the fruit fly, Drosophila melanogaster, has become an invaluable model in which to dissect the neurological basis of conserved complex behaviors. Here, I characterized the respective roles of two genes in alcohol response and sleep behavior. Steroid hormones profoundly influence behavioral response to alcohol, yet the role of unconventional non-genomic steroid signaling in this process is unknown. I discovered that Drosophila DopEcR, a G-protein coupled receptor (GPCR) activated by dopamine or the major insect steroid hormone ecdysone, plays a critical role in ethanol-induced behaviors. DopEcR mutants took longer to sedate when exposed to ethanol vapor, and post-eclosion expression of DopEcR-RNAi phenocopied mutant resistance. DopEcR was necessary in particular neuronal subsets, including cholinergic and peptidergic neurons, and promoted ethanol sedation by suppressing epidermal growth factor/extracellular signal-regulated kinase signaling. In adult flies, ecdysone negatively regulated DopEcR-mediated ethanol-induced sedation. We also found that DopEcR inhibits ethanol-induced locomotion, a conserved dopaminergic behavior. Together, these findings provide novel insight into how an unconventional steroid GPCR interacts with multiple downstream signaling cascades to fine tune behavioral response to alcohol. Despite an established link between epilepsy and sleep behavior, it remains unclear how epileptogenic mutations affect sleep and seizure susceptibility. To address this, I examined the rest/wake behavior of two fly models of epilepsy with paralytic voltage-gated sodium channel mutations known to cause human generalized epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS). GEFS+ and DS flies display heat-induced seizure susceptibility, but at normal temperatures I found that both mutants had exaggerated nighttime sleep behavior. GEFS+ sleep was more resistant to pharmacologic and genetic reduction of GABA transmission as compared to control’s response. This finding is consistent with augmented GABAergic suppression of wake-promoting pigment-dispersing factor (PDF) neurons in GEFS+ mutants. Contrastingly, DS sleep was almost completely resistant to pharmacologic GABA reduction, suggesting that PDF neurons are incapable of functioning despite disinhibition. The sleep of both GEFS+ and DS flies was largely suppressed, but not eliminated, by scotophase light, highlighting the importance of light stimulus and circadian signals in the manifestation of their phenotypes. Following sleep deprivation, GEFS+ and DS mutants failed show to homeostatic rebound. Sleep loss also unexpectedly reduced the seizure susceptibility of GEFS+ flies. This study revealed the sleep architecture of Drosophila voltage-gated sodium channel mutants and provides a unique platform in which to further study the sleep/epilepsy relationship.

About 5% to 10% of epileptic patients suffer from Juvenile Myoclonic Epilepsy (JME), which is characterized by spasms of the arms, ataxia (uncoordinated movements), and general tonic-clonic seizures. In a recent study, a group of patients with myoclonic epilepsy was found to harbor mutations in the PRICKLE1 and PRICKLE2 genes. This suggested that PRICKLE genes might be linked to epilepsy, and given that PRICKLE is highly evolutionarily conserved (including in fruit flies), we decided to use Drosophila in order to determine, first, whether flies with prickle mutations were seizure-prone, and if so, to then use the powerful genetic tools of Drosophila to elucidate the underlying mechanism of the prickle-associated epilepsy. In this work, we show that mutation of the pksple isoform (one of the two adult prickle isoforms in flies) lowers the seizure threshold in the mutant flies (resulting in seizure activity), while mutation of the other adult isoform, pkpk, had no effect. This was demonstrated through both behavioral assays (where the pksple mutant flies showed a reduction in recovery of climbing behavior after being subjected to mechanical stimulation while the pkpk mutant flies did not) as well as electrophysiological analysis (where pksple mutants were shown to be hyperexcitable after electrical stimulation, while the pkpk allele showed no change in spiking activity). We demonstrated that the underlying mechanism of the hyperexcitability seen in the pksple flies was due to enhanced anterograde transport on microtubule (MT) tracks in neurons, the main route for transport in neurons, which could be suppressed by reducing the dose of either of two Kinesin motor proteins, the motors involved in anterograde transport in neurons. On the other hand, the pkpk mutants showed the reverse effect, exhibiting a significant reduction in vesicle transport dynamics. We showed that microtubule polarity could be partially reversed by tipping the balance of the pk isoforms similar to what is seen in the pkpk mutants (such that a large percentage of MTs now had their plus ends oriented towards the cell body, which is extremely rare in axons), suggesting that the vesicle transport defects seen in the pkpk mutants might be due to mixed polarity of MTs. Next, we showed that the seizure-prone pksple mutants, but not the pkpk mutants, exhibited a myoclonic form of epilepsy, as well as abnormal walking patterns and uncoordinated movements, paralleling the ataxia phenotype seen in the epileptic patients with PRICKLE mutations. These data suggest that the primary aspects of the epilepsy-ataxia syndrome seen in patients with PRICKLE mutations are recapitulated in flies, which underscores the utility of using the fruit fly genetic system to model this disorder. Finally, our preliminary results suggest that the pk alleles have different effects on neuronal morphology due to changes in sizes of terminal boutons at the neuromuscular junction (NMJ) in larvae. These data suggest that pk is having a direct effect on synaptic formation and likely function. In conclusion, by using our Drosophila model system, we were able to link prickle mutations to epilepsy as well as identify the cellular mechanism of the prickle-associated epilepsy, a novel epilepsy mechanism previously associated with neurodegeneration. To our knowledge, this is the first example of a gene that, when mutated, will cause seizures in flies, zebrafish, mice, and humans, indicating that the role of prickle in controlling seizure activity is remarkably conserved in animals. Significantly, since about one third of patients with epilepsy do not respond to current AEDs, our fly model and the techniques we have developed will enable us to conduct drug screens for testing potential chemical compounds as new AEDs.

No description available.

No description available.

En la presente investigación, se busca conocer las percepciones sociales de los pacientes adultos con epilepsia. Se realizó un estudio cualitativo fenomenológico. La muestra estuvo conformada por 8 participantes (6 varones y 2 mujeres) entre los 22 y 60 años, diagnosticados con epilepsia. Se realizó una entrevista semiestructurada para la cual se elaboraron nueve preguntas. Se construyó una guía con cuatro ejes: a) la percepción del sí mismo de la persona con epilepsia, b) percepción de las personas con epilepsia sobre sus relaciones interpersonales, c) percepciones de epilepsia en su interacción socio laboral d) percepción de la epilepsia sobre su atención y acceso a los servicios de salud. Los hallazgos demuestran que los participantes presentan auto concepto negativo frente a su condición. Perciben que son incluidos socialmente, no obstante, tienden a aislarse. Algunos consideran el cuidado de su clan como sobreprotección familiar, y otros participantes lo valoran como soporte familiar. Los participantes manifiestan ser capaces de reconocer los posibles potenciales episodios desencadenantes de su enfermedad. / In the present investigation, we seek to know the social perceptions of adult patients with epilepsy. A qualitative phenomenological study was carried out. The sample consisted of 8 participants (6 men and 2 women) between 22 and 60 years old, diagnosed with epilepsy. A semi-structured interview was conducted for which nine questions were prepared. A guide was built with four axes: a) the perception of the self of the person with epilepsy, b) perception of people with epilepsy about their interpersonal relationships, c) perceptions of epilepsy in their social-professional interaction and d) perception of epilepsy about their attention and access to Health services. The findings show that the participants have a negative self-concept about their condition. They perceive that they are included socially, however, they tend to isolate themselves. Some consider the care of their clan as family overprotection, and other participants value it as family support. The participants declare that they are able to recognize the possible potential episodes that trigger their illness. / Tesis

Defining language lateralisation is important to minimise morbidity in patients treated surgically for temporal lobe epilepsy (TLE). Functional magnetic resonance imaging (fMRI) offers a promising, non-invasive, alternative strategy to the Wada test. Here, fMRI has been used to study healthy controls and patients with TLE in order (i) to define language-related activation patterns and their reproducibility; (ii) to compare lateralisation determined by fMRI with that from the Wada test; and (iii) to explore the usefulness of multiple fMRI language paradigms. 18 healthy controls (12 right-handed and 6 left-handed) and 24 pre-operative TLE patients (19 right-handed: 12 left-TLE, 7 right-TLE; 5 left-handed: 2 right-TLE, 3 left-TLE) were studied using fMRI. Four fMRI language paradigms used: phonetic and semantic fluency, and the naming of living and non-living things. The data for all 4 tasks were acquired during a single scanning session on two occasions. All patients also underwent Wada testing. In patients and controls, phonetic and semantic fluency tasks were robustly activating and strongly lateralising. Quantified language-related lateralisation from fMRI verbal fluency data was highly reproducible and concordant with the lateralisation of the Wada test. Both fluency tasks identified patients with atypical language lateralisation, including 4/12 right-handed patients with left-TLE and 4/5 left-handed TLE patients, regardless of the side of epileptic focus. In comparison, the two confrontational naming tasks were not strongly lateralising and did not reliably agree with Wada lateralisation in either 12 right-handed controls or 19 right-handed patients with TLE. However, there was a difference in the pattern of fMRI activation in right-handed pat ients with left-TLE. Left-TLE patients had a more right lateralised network of activation when naming living things relative to non-living things, suggesting that some patients may be at risk of a category specific naming decline for non-living things after left anterior temporal lobectomy. These results demonstrate that non-invasive fMRI measures of languagerelated lateralisation may provide a practical and reliable alternative to invasive testing for pre-surgical language lateralisation in patients with TLE. The high proportion of TLE patients showing atypical language lateralisation suggests considerable plasticity of language representation in the brains of patients with intractable TLE.

Degree awarded (2004): PhDBmS, Neuroscience Program, George Washington University / Glutamate is critically important as an excitatory neurotransmitter in the central nervous system. Increasing evidence suggests additional signaling roles for glutamate in cell proliferation and migration in normal and oncogenic states. Recently, glutamate release from glioma cells has been shown to increase tumor growth in vivo. To investigate the mechanism of glutamate enhancement of tumor growth, we investigated the effect of glutamate on tumor cell proliferation, invasion, and glioma-induced cell death. Here we demonstrate that glutamate enhances tumor growth via increasing tumor cell proliferation and inducing excitotoxic death of cells surrounding the solid tumor mass, thereby facilitating tumor expansion. The evidence that glutamate enhances tumor growth suggests that regulating extracellular levels of glutamate may restrict tumor growth. In the normal brain, extracellular glutamate levels are maintained by a family of glutamate transporters. To investigate the therapeutic potential of regulating extracellular glutamate concentrations on tumor growth, we utilized a transgenic mouse model of EAAT2 glutamate transporter overexpression. In this report, we demonstrate that increased glutamate transport limits tumor growth in vivo and provides protection against glioma-associated neuronal cell death. In addition, seizure activity, often associated with the presence of a CNS tumor, is attenuated in transgenic mice overexpressing the glutamate transporter, EAAT2. These findings suggest that glutamate transporters may provide a new therapeutic target for limiting tumor expansion and secondary epileptogenesis. / Advisory Committee: Dr. Margaret Sutherland (Chair), Dr. Steven Patierno (Chair), Dr. Tim Hales, Dr. Vincent Chiappinelli, Dr. Linda Werling, Dr. Frances Noonan

Thesis (M. Phil.)--University of Hong Kong, 2006. / Also available in print.