Most (99%) mitochondrial proteins are nuclear-encoded and must be imported into mitochondria. Deficits in mitochondrial protein import (MPI) affect mitochondrial function and can cause neurodegenerative diseases. I hypothesized that MPI was regulated by iCa2+. In differentiated PC12 cells, treatment with the Ca2+ ionophore (A23187; 24h, 0.15uM) increased iCa2+, ROS generation and promoted neurite outgrowth. Western blot and flow cytometry in live cells showed that A23187 increased levels of mitochondrial proteins; mtHSP70 and mtGFP in mitochondria and autoradiography confirmed that A23187 increased the import of mtGFP. A23187 also slowed intramitochondrial mtGFP degradation. Increased MPI was not associated with mitochondrial biogenesis, but appeared partially dependent on cAMP. In rat cortical neurons, mtHSP70 also increased after A23187 treatment. These results show that, in neurons, increased iCa2+ can regulate MPI. Further, increased iCa2+ can slow intramitochondrial protein degradation. These results indicate that MPI is labile and may be altered in response to neuronal activity.

Our work on RGMb demonstrates a clear and new role in the developing chick visual system. RGMb is expressed in distinct areas of the developing visual system: retinal ganglion cells (RGCs) of the retina, which are the only cells in the visual system that extend axons to the brain, as well as newly differentiated neuronal cells within the optic tectum (OT), the primary target of RGC axons. Knockdown of RGMb in RGCs at embryonic day 2 (E2) resulted in aberrant axon projection at E17, indicating that RGMb is required for axon development. Furthermore, knockdown of RGMb in the optic tectum at E5 resulted in disrupted cellular migration at E9, demonstrating that RGMb is involved in correct cell migration. Lastly, we demonstrated that RGMb binds to the Fibronectin III (3,4) domain of Neogenin, which provides a basis for determining the mechanism through which RGMb exerts its biological effects.

The structure of afferent neurons is pseudounipolar. Studies suggest that they relay action potentials (APs) to both directions of the T-junctions to reach the cell body and the spinal cord. Moreover, the somata are electrically excitable and shown to be able to transmit the signals to associated satellite cells. Our study demonstrates that this transmission can go further and pass onto passive neighbouring somata, if they are in direct contact with same satellite cells. The neurons activate the satellite cells by releasing ATP. This triggers the satellite cells to exocytose acetylcholine to the neighbouring neurons. In addition, the ATP inhibits the nicotinic receptors of the neurons by activating P2Y receptors and initiating the G-protein-mediated pathway, thus reducing the signals that return to the neurons that initiated the signals. This “sandwich synapse” represents a unique pathway by the ectopic release between the somata and the satellite cells.

Most (99%) mitochondrial proteins are nuclear-encoded and must be imported into mitochondria. Deficits in mitochondrial protein import (MPI) affect mitochondrial function and can cause neurodegenerative diseases. I hypothesized that MPI was regulated by iCa2+. In differentiated PC12 cells, treatment with the Ca2+ ionophore (A23187; 24h, 0.15uM) increased iCa2+, ROS generation and promoted neurite outgrowth. Western blot and flow cytometry in live cells showed that A23187 increased levels of mitochondrial proteins; mtHSP70 and mtGFP in mitochondria and autoradiography confirmed that A23187 increased the import of mtGFP. A23187 also slowed intramitochondrial mtGFP degradation. Increased MPI was not associated with mitochondrial biogenesis, but appeared partially dependent on cAMP. In rat cortical neurons, mtHSP70 also increased after A23187 treatment. These results show that, in neurons, increased iCa2+ can regulate MPI. Further, increased iCa2+ can slow intramitochondrial protein degradation. These results indicate that MPI is labile and may be altered in response to neuronal activity.

Our work on RGMb demonstrates a clear and new role in the developing chick visual system. RGMb is expressed in distinct areas of the developing visual system: retinal ganglion cells (RGCs) of the retina, which are the only cells in the visual system that extend axons to the brain, as well as newly differentiated neuronal cells within the optic tectum (OT), the primary target of RGC axons. Knockdown of RGMb in RGCs at embryonic day 2 (E2) resulted in aberrant axon projection at E17, indicating that RGMb is required for axon development. Furthermore, knockdown of RGMb in the optic tectum at E5 resulted in disrupted cellular migration at E9, demonstrating that RGMb is involved in correct cell migration. Lastly, we demonstrated that RGMb binds to the Fibronectin III (3,4) domain of Neogenin, which provides a basis for determining the mechanism through which RGMb exerts its biological effects.

Noise-like activity (NLA) refers to spontaneous subthreshold fluctuations in membrane potential. In this thesis, we examine the role that synaptic channel fluctuations play in contributing to NLA by comparing a detailed biophysical model to experimental data from whole-intact hippocampal interneurons. To represent the contribution from synaptic channel fluctuations, we switch the synapses in the model from traditional to Markovian formalisms and demonstrate statistically relevant increases the standard deviation; power-law scaling exponent; and power spectral density in the 5-100 Hz and 1-5 kHz ranges. However, while synaptic channel fluctuations have a definite effect, we found that they were significantly more subtle than the synaptic response to network activity. This indicates that synaptic channel fluctuations do indeed play a significant role in subthreshold noise, but, overall, synaptic NLA is dominated by the synaptic response to presynaptic network activity.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Sensory-motile cells fulfill various biological functions ranging from immune activity or wound healing to the formation of the highly complex nervous systems of vertebrates. In the case of neurons, a dynamic structure at the tip of outgrowing processes navigates towards target cells or areas during the generation of neural networks. These fan shaped growth cones are equipped with a highly complex molecular machinery able to detect various external stimuli and to translate them into directed motion. Receptor and adhesion molecules trigger signaling cascades that regulate the dynamics of an internal polymeric scaffold, the cytoskeleton. It plays a crucial role in morphology maintenance as well as in the generation and distribution of growth cone forces. The two major components, actin and microtubules (MTs) connect on multiple levels through interwoven biochemical and mechanical interactions. Actin monomers assemble into semiflexible filaments (F-actin) which in turn are either arranged in entangled networks in the flat outer region of the growth cone (lamellipodium) or in radial bundles termed filopodia. The dynamic network of actin filaments extends through polymerization at the front edge of the lamellipodium and is simultaneously moving towards the center (C-domain) of the growth cone. This retrograde flow (RF) of the actin network is driven by the polymerizing filaments themselves pushing against the cell membrane and the contractile activity of motor proteins (myosins), mainly in the more central transition zone (T-zone). Through transmembrane adhesion molecules, a fraction of the retrograde flow forces is mechanically transmitted to the cellular substrate in a clutch-like mechanism generating traction and moving the GC forward. MTs are tubular polymeric structures assembled from two types of tubulin protein subunits. They are densely bundled in the neurite and at the growth cone “neck” (where the neurite opens out into the growth cone) they splay apart entering the C-domain and more peripheral regions (P-domain). Their advancement is driven by polymerization and dynein motor protein activity. The two subsystems, an extending array of MTs and the centripetal moving actin network are antagonistic players regulating GC morphology and motility. Numerous experimental findings suggest that MTs pushing from the rear interact with actin structures and contribute to GC advancement. Nevertheless, the amount of force generated or transmitted through these rigid structures has not been investigated yet. In the present dissertation, the deformation of MTs under the influence of intracellular load is analyzed with fluorescence microscopy techniques to estimate these forces. RF mechanically couples to MTs in the GC periphery through friction and molecular cross-linkers. This leads to MT buckling which in turn allows the calculation of the underlying force. It turns out that forces of at least act on individual MT filaments in the GC periphery. Compared to the relatively low overall protrusion force of neuronal GCs, this is a substantial contribution. Interestingly, two populations of MTs buckle under different loads suggesting different buckling conditions. These could be ascribed to either the length-dependent flexural rigidity of MTs or local variations in the mechanical properties of the lamellipodial actin network. Furthermore, the relation between MT deformation levels and GC morphology and advancement was investigated. A clear trend evolves that links higher MT deformation in certain areas to their advancement. Interactions between RF and MTs also influence flow velocity and MT deformation. It is shown that transient RF bursts are related to higher MT deformation in the same region. An internal molecular clutch mechanism is proposed that links MT deformation to GC advancement. When focusing on GC dynamics it is often neglected that the retraction of neurites and the controlled collapse of GCs are as important for proper neural network formation as oriented outgrowth. Since erroneous connections can cause equally severe malfunctions as missing ones, the pruning of aberrant processes or the transient stalling of outgrowth at pivotal locations are common events in neuronal growth. To date, mainly short term pausing with minor cytoskeletal rearrangements or the full detachment and retraction of neurite segments were described. It is likely that these two variants do not cover the full range of possible events during neuronal pathfinding and that pausing on intermediate time scales is an appropriate means to avoid the misdetection of faint or ambiguous external signals. In the NG108-15 neuroblastoma cells investigated here, a novel type of collapse was observed. It is characterized by the degradation of actin network structures in the periphery while radial filopodia and the C-domain persist. Actin bundles in filopodia are segmented at one or multiple breaking points and subsequently fold onto the edge of the C-domain where they form an actin-rich barrier blocking MT extension. Due to this characteristic, this type of collapse was termed fold collapse. Possible molecular players responsible for this remarkable process are discussed. Throughout fold collapse, GC C-domain area and position remain stable and only the turnover of peripheral actin structures is abolished. At the same time, MT driven neurite elongation is hindered, causing the GC to stall on a time scale of several to tens of minutes. In many cases, new lamellipodial structures emerge after some time, indicating the transient nature of this collapse variant. From the detailed description of the cytoskeletal dynamics during collapse a working model including substrate contacts and contractile actin-myosin activity is derived. Within this model, the known and newly found types of GC collapse and retraction can be reduced to variations in local adhesion and motor protein activity. Altogether the results of this work indicate a more prominent role of forward directed MT-based forces in neuronal growth than previously assumed. Their regulation and distribution during outgrowth has significant impact on neurite orientation and advancement. The deformation of MT filaments is closely related to retrograde actin flow which in turn is a regulator of edge protrusion. For the stalling of GCs it is not only required that actin dynamics are decoupled from the environment but also that MT pushing is suppressed. In the case of fold collapse, this is achieved through a robust barrier assembled from filopodial actin bundles.

No description available.

This research focuses on the nanoscopic investigation of the three-dimensional surface morphology of the neural growth cones from the snail Helisoma trivolvis, and InN and InGaN semiconductor material systems using Atomic Force Microscopy (AFM). In the analysis of the growth cones, the results obtained from AFM experiments have been used to construct a 3D architecture model for filopodia. The filopodia from B5 and B19 neurons have exhibited different tapering mechanisms. The volumetric analysis has been used to estimate free Ca2+ concentration in the filopodium. The Phase Contrast Microscopy (PCM) images of the growth cones have been corrected to thickness provided by AFM in order to analyze the spatial refractive index variations in the growth cone. AFM experiments have been carried out on InN and InGaN epilayers. Ternary InGaN alloys are promising for device applications tunable from ultraviolet (Eg[GaN]=3.4 eV) to near-infrared (Eg [InN]=0.7 eV). The real-time optical characteristics and ex-situ material properties of InGaN epilayers have been analyzed and compared to the surface morphological properties in order to investigate the relation between the growth conditions and overall physical properties. The effects of composition, group V/III molar ratio and temperature on the InGaN material characteristics have been studied and the growth of high quality indium-rich InGaN epilayers are demonstrated.