Expanding the mutational spectrum and investigating the pathophysiology of GOSR2 mediated progressive myoclonus epilepsy

In this PhD thesis I summarize my research into the genetics and pathophysiology of progressive myoclonus epilepsy (PME) associated with mutations in GOSR2. This disorder is characterized by early disease onset with ataxia around 3 years of age, followed by development of cortical myoclonus, generalized epilepsy and a rapid deterioration of motor function. Upon beginning my PhD, only one homozygous GOSR2 mutation – c.430G>T (p.G144W) – had been shown to cause PME. Furthermore, because GOSR2 encodes a Golgi SNARE protein (termed Membrin) that mediates ER-to-Golgi trafficking in every cell of the human body, it was an unresolved mystery how this mutation gives rise to a largely selective neuronal disorder. I first describe my discovery of the novel c.491-493delAGA (p.K164del) GOSR2 mutation in a PME patient who also carried the previously described c.430G>T variant in the compound heterozygous state. Overall, the clinical phenotype of this patient was remarkably consistent with previous cases, although her disease course appeared milder. My finding thus expanded the phenotypes and genotypes linked to this disorder, thus providing an additional tool to investigate the underlying disease mechanisms. In the subsequent chapters I summarize our attempts to unravel why the nervous system is selectively affected in GOSR2-PME. To this end I examined how pathogenic Membrin mutations impacted ER-to-Golgi trafficking in patient-derived fibroblasts, and developed novel Drosophila models of GOSR2-PME to study neuronal pathophysiology. Intriguingly, while ER-to-Golgi trafficking was remarkably preserved in G144W mutant Membrin fibroblasts, neuronal integrity was severely disturbed in GOSR2- PME model Drosophila, where dendrites were significantly shorter. Neurons have special secretory demands owing to their very large surface area, and hence appear selectively vulnerable to partial loss of function mutations in Membrin. Thus, the results presented in this thesis provide a possible explanation for the nervous system specificity of GOSR2-PME

High flavonoid accompanied with high starch accumulation triggered by nutrient starvation in bioenergy crop duckweed (Landoltia punctata)

Background: As the fastest growing plant, duckweed can thrive on anthropogenic wastewater. The purple-backed duckweed, Landoltia punctata, is rich in starch and flavonoids. However, the molecular biological basis of high flavonoid and low lignin content remains largely unknown, as does the best method to combine nutrients removed from sewage and the utilization value improvement of duckweed biomass. Results: A combined omics study was performed to investigate the biosynthesis of flavonoid and the metabolic flux changes in L. punctata grown in different culture medium. Phenylalanine metabolism related transcripts were identified and carefully analyzed. Expression quantification results showed that most of the flavonoid biosynthetic transcripts were relatively highly expressed, while most lignin-related transcripts were poorly expressed or failed to be detected by iTRAQ based proteomic analyses. This explains why duckweed has a much lower lignin percentage and higher flavonoid content than most other plants. Growing in distilled water, expression of most flavonoid-related transcripts were increased, while most were decreased in uniconazole treated L. punctata (1/6 x Hoagland + 800 mg center dot L-1 uniconazole). When L. punctata was cultivated in full nutrient medium (1/6 x Hoagland), more than half of these transcripts were increased, however others were suppressed. Metabolome results showed that a total of 20 flavonoid compounds were separated by HPLC in L. punctata grown in uniconazole and full nutrient medium. The quantities of all 20 compounds were decreased by uniconazole, while 11 were increased and 6 decreased when grown in full nutrient medium. Nutrient starvation resulted in an obvious purple accumulation on the underside of each frond. Conclusions: The high flavonoid and low lignin content of L. punctata appears to be predominantly caused by the flavonoid-directed metabolic flux. Nutrient starvation is the best option to obtain high starch and flavonoid accumulation simultaneously in a short time for biofuels fermentation and natural products isolation

Neurodegeneration cell per cell

The clinical definition of neurodegenerative diseases is based on symptoms that reflect terminal damage of specific brain regions. This is misleading as it tells little about the initial disease processes. Circuitry failures that underlie the clinical symptomatology are themselves preceded by clinically mostly silent, slowly progressing multicellular processes that trigger or are triggered by the accumulation of abnormally folded proteins such as Aβ, Tau, TDP-43, and α-synuclein, among others. Methodological advances in single-cell omics, combined with complex genetics and novel ways to model complex cellular interactions using induced pluripotent stem (iPS) cells, make it possible to analyze the early cellular phase of neurodegenerative disorders. This will revolutionize the way we study those diseases and will translate into novel diagnostics and cell-specific therapeutic targets, stopping these disorders in their early track before they cause difficult-to-reverse damage to the brain