Age-dependent decline in Kv4 channels, underlying molecular mechanisms, and potential consequences for coordinated motor function

2019 Spring.Includes bibliographical references.The voltage-gated potassium channel, Kv4, is widely expressed in the central nervous system and it is responsible for a highly conserved rapidly inactivating A-type K+ current. Kv4 channels play a role in the regulation of membrane excitability, contributing to learning/memory and coordinated motor function. Indeed, recent genetic and electrophysiological studies in Drosophila have linked Kv4 A-type currents to repetitive rhythmic behaviors. Because a deterioration in locomotor performance is a hallmark of aging in all organisms, we were interested in examining the effects of age on Kv4/Shal channel protein. In this dissertation, I use Drosophila as a model organism to characterize an age-dependent decline in Kv4/Shal protein levels that contributes to the decline in coordinated motor performance in aging flies. Our findings suggest that accumulation of hydrogen peroxide (H2O2) is amongst the molecular mechanisms that contribute to the age-dependent decline of Kv4/Shal. We show that an acute in vivo H2O2 exposure to young flies leads to a decline of Kv4/Shal protein levels, and that expression of Catalase in older flies results in an increase in levels of Kv4/Shal and improved locomotor performance. We also found that the scaffolding protein SIDL plays a role in maintaining Kv4/Shal protein levels and that SIDL mRNA declines with age, suggesting that an age-dependent loss of SIDL may also lead to Kv4/Shal loss. In behavioral studies, we found that a knockdown of SIDL resulted in a lethal phenotype, leading to a large decline in Drosophila eclosion rates, an event that requires coordinated peristaltic motions. Expression of SIDL or Kv4/Shal in this SIDL knockdown genetic background resulted in a partial rescue; these results are consistent with a model in which SIDL and Kv4/Shal play a role in coordinated peristaltic motions and are required for successful eclosion. The results presented in this dissertation provide new insight into the possible molecular mechanisms that underlie an age-dependent decline in Kv4/Shal protein. We identify two contributing factors: 1) ROS accumulation, and 2) the interacting protein SIDL. Our data also suggests that this age-dependent decline in Kv4/Shal levels is likely to be conserved across species, at least in some brain regions. Because Kv4/Shal channels have been implicated in the regulation of long-term potentiation and in repetitive rhythmic behaviors, the loss of Kv4/Shal may contribute to the age-related decline in learning/memory and motor function

THE ROLE OF NADPH OXIDASE IN NEURITE OUTGROWTH AND ZEBRAFISH NEURODEVELOPMENT

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are a family of enzymes that produce reactive oxygen species (ROS). The first NOX enzyme was discovered in leukocytes and associated with host defense in the immune system. Subsequent findings of ROS production in non-immune cells led to the identification of six additional NOX isoforms, and opened new avenues for research into NOX-mediated cellular functions. Since then, NOX-derived ROS have been found to be involved in a tremendous number of cell signaling pathways. Of particular interest is the well-established function of NOX-derived ROS in signaling pathways that drive cytoskeletal rearrangements and motility in several cell types. Our lab is interested in the highly motile neuronal growth cone that guides axonal growth during neurodevelopment and regeneration. Others have reported that inhibition of NOX enzymes during development causes a decrease in the size of some brain areas, and NOX deficiencies in humans are correlated with diminished cognitive function. Despite the fact that NOX activity is necessary for some cell motility in non-neuronal cells and a loss of NOX function during development has impacts on brain structure, it is still unclear what role NOX plays in axonal growth and guidance or the establishment of connections in the central nervous system

The actin-binding protein Drebrin and its implications for Alzheimer's Disease using the model organism C. elegans

Patients of Alzheimer’s Disease (AD) showed reduced levels of the actin-binding protein Drebrin in their neurons. The here presented work was set out to analyse the interaction between Drebrin and the disease-associated peptide Amyloid-β. To analyse the interaction in vivo two novel models were designed, employing the nematode C. elegans. The Amyloid-β pathology was modelled by overexpressing the disease causing peptide pan-neuronally and employing a genetic sub-stoichiometric labelling method to be able to follow the aggregation in vivo and in situ in a non-invasive manner. A second model, expressing human Drebrin pan-neuronally was generated to analyse Drebrin stability, localization and phosphorylation as well as analysing the effect of Drebrin overexpression on the nematodes’ vitality and fitness. The third project combined both generated models to obtain a genetic cross expressing Aβ(1-42) and Drebrin simultaneously. This model was sought to study the interaction between Aβ(1-42) and Drebrin. I could show, that Aβ(1-42) aggregates with the progression of ageing and exhibits multiple disease phenotypes that can be correlated to observations obtained in murine neurons as well as observations of AD patients’ brain tissues. Furthermore, I observed, that a distinct subset of head neurons of the anterior ganglion, the IL2 neurons, exhibits the first aggregates and that a cell-type specific suppression of Aβ(1-42) in IL2 neurons could delay the disease onset. Drebrin was observed to be regulated by phosphorylation at Serine-647 by Ataxia telangiectasia mutated kinase and render nematodes more resistant towards chronic oxidative stress. The genetic cross of Aβ(1-42) and Drebrin unravelled that overexpression of Drebrin can ameliorate Aβ(1-42) aggregation and toxicity and that this beneficial effect is dependent on phosphorylation of Drebrin-S647

Impact of ionising radiation on adult neurogenesis: physiological and cellular effects of low and moderate doses of ionising radiation in neural differentiation distinguished by differentiation phase

Comprehensive follow-up of brain cancer patients and re-evaluation of medical data revealed a frightening correlation between low and moderate doses of ionising irradiation and induced cognitive dysfunctions. The particular radio sensitivity of adult neurogenesis within the adult brain is suggested as the major contributor in the underlying pathogenesis. Adult neurogenesis describes the differentiation of neural stem cells to mature neurons in the adult brain. Neural differentiation can be separated in three main phases, early progenitor phase, fate specification phase and cell maturation phase. The cellular base of neural differentiation provides a restricted stem cell pool, out of self-renewing neural stem cells (NSCs). To investigate the impact of low and moderate doses of ionising radiation (IR) on adult neurogenesis we intended to distinguish the differentiation process in its dynamic subpopulations, represented in the individual differentiation phases. Based on ES-derived NSCs we established and characterised a 2D-model system, reflecting the three differentiation phases, of adult neurogenesis. In the first part of this work we characterised the differentiation of the J1 NSC model system concerning specific marker expression, morphology and functional differentiation markers. The broad characterisation revealed that the J1 model system reflects each of the three differentiation phases, i.e. early progenitor, fate specification and cell maturation in adult neurogenesis on the intra- and intercellular as well as on the functional level in a synchronised, time dependent differentiation. We used the synchronous differentiation of the J1 model system to discriminate the individual differentiation phases by time. In the second part of this work we analysed the individual radio sensitivity of the distinct differentiation phases. Addressing issues concerning radiotherapy bystander and diagnostic doses, we used low to moderate x-ray doses between 0.25 and 2Gy. To estimate the distinct radio sensitivity of NSCs and the three differentiation phases, we measured the reduction in vitality of the separated subpopulations and estimated the individual LD50 of each differentiation phase. NSCs, as well as all three differentiation phases, show an individual radio sensitivity significantly different to the other subpopulations. The proliferative subpopulations, NSCs and early progenitors showed the highest radio sensitivity, whereas the final differentiation phase, cell maturation, showed the lowest. In further analyses we investigated the mechanisms responsible for the IR induced reductions in the individual subpopulations. Within the postmitotic differentiation phases, fate specification and cell maturation, radiation induced apoptosis is the underlying mechanism. In the proliferative subpopulations, the reduction in the number of cells is predominantly mediated by reduced proliferation in NSCs and induced apoptosis in early progenitors. To investigate the effect of IR on individual differentiation phases further, we irradiated the three defined differentiation phases and analysed the characteristic differentiation properties of the particular differentiation phase by functional, morphological and histochemical markers post IR. Early progenitors, the first subpopulation within the neural differentiation, are affected in their proliferation, but the migration of the surviving cells is not affected by low dose IR, neither is the entry into the postmitotic status. In the next step we analysed J1 cells irradiated in the fate specification phase. The fate determination of the surviving cell population showed a reduced percentage of future neurons post ionising radiation compared to unirradiated samples. The terminal differentiation phase revealed a broad modulation in the phase specific characteristic properties induced by low dose radiation, determined in altered neuronal architecture and a lower density of synaptic markers in neuroblasts as well as a reduced density of the voltage-gated potassium channels KV1.1. Regarding the functional level we found a long-lasting stagnation in the excitability of the in cell maturation phase irradiated cultures, reflected by a reduced firing rate, decreased coordination in the activity pattern and a loss in spike synchrony. In the last part of this work we investigated whether the properties of the self-renewing NSCs were also affected by IR. In previous work Dr. Bastian Roth determined that functional characteristics of J1 NSCs are modified post low dose IR, mediated via alterations in K+ channel currents. In continuative experiments we investigated if low dose IR leads to changes in the morphological and immunohistological characteristic within the J1 NSC population. The follow up of the irradiated NSCs revealed a highly significant increase of the differentiation marker doublecortin (DCX) within the self-renewing population. By using K+ channel blockers during radiation, we could inhibit the increase in DCX positive cells post low dose IR. In summary we found radiation induced modulations in the phase specific properties of each subpopulation, represented by NSCs and the three differentiation phases, in the third and fourth part of this work. In conclusion, depending on the differentiation phase, we could identify several specific physiological and cellular effects of low and moderate doses of IR during neural differentiation in a ES-derived neural stem cell line