The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network

Early endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150(glued) component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

The role of the cortical occipital spike in photosensitive epilepsy

Chapters one to three are an introduction to photosensitive epilepsy, electroencephalography (EEG) and the magnocellular and parvocellular visual pathways. Photoparoxysmal response (PPR) are strongly associated with photosensitive epilepsy. Chapters four to nine investigated whether occipital spikes were associated with PPR and hence with photosensitive epilepsy. The chapters investigated whether the response types showed similar dependence on stimulus characteristics using EEG. Chapters four and five found that occipital spikes and PPR showed different dependence on colour and luminance contrast. The differences were consistent with the magnocellular pathway mediating occipital spikes and the pavocellular pathway mediating PPR. The study in chapter eight found that monocular occlusion had a significantly greater effect on PPR than on occipital spikes, which is further evidence against an association between the two types of response. Chapters six and seven showed that occipital spikes and PPR had similar optimum spatial and temporal frequencies. Chapter nine showed that both response types could be generated via stimulation of the periphery of the retina. However, these three chapters are not strong evidence of an association, as the results do not contradict the theory that the responses are generated via different pathways. The magnocellular and pavocellular pathways have similar optimum temporal and spatial frequencies and both are present in the periphery. In chapter ten, magnetoencephalography was used to estimate the source of activity underlying the components of the VEP and occipital spike. Changes in the amplitude and latency in the components of the normal VEP are associated with epilepsy. However, the source underlying the occipital spikes was not related to that underlying the components of the VEP so this is also removed as a source of evidence for an association between occipital spikes and photosensitive epilepsy.

An investigation of the pressaccidic spike potential

A large negative spike potential, which is closely related to the onset of saccadic eyemovements, can be recorded from electrodes adjacent to the orbits. This potential, thepresaccadic spike potential, has often been regarded as an artefact related to eyemovement recordings and little work has been performed to establish its normal waveformand parameters. A positive spike potential, exactly coincident with the frontal negativespike, has also been recorded from electrodes positioned over the posterior scalp andthere has been some debate regarding any possible relationship between the twopotentials. The frontal spike potential has been associated with motor unit activity in theextraocular muscles prior to the saccade. This thesis investigates both the large anteriorand smaller posterior spike potentials and relates these recordings to the saccadic eyemovements associated with them. The anterior spike potential has been recorded from normal subjects to ascertain its normallatency and amplitude parameters for both horizontal and vertical saccades. A relationshipbetween saccade size and spike potential amplitude is described, the spike potentialamplitude reducing with smaller saccades. The potential amplitude also reduces withadvancing age. Studying the topographical distribution of the spike potential across thescalp shows the posterior spike activity may arise from potential spread of the larger frontalspike potential. Spike potential recordings from subjects with anomalous eye movements further implicate the extraocular muscles and their innervation in the generation of the spike potential. These recordings indicate that the spike potential may have some use as a clinical recording from patients with disease conditions affecting either their extraocular muscles or the innervational pathways to these muscles. Further recordings of the potential are necessary, however, to determine the exact nature of the changes which may occur with such conditions.

SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.

Spike firing and IPSPs in layer V pyramidal neurons during beta oscillations in rat primary motor cortex (M1) in vitro

Beta frequency oscillations (10-35 Hz) in motor regions of cerebral cortex play an important role in stabilising and suppressing unwanted movements, and become intensified during the pathological akinesia of Parkinson's Disease. We have used a cortical slice preparation of rat brain, combined with concurrent intracellular and field recordings from the primary motor cortex (M1), to explore the cellular basis of the persistent beta frequency (27-30 Hz) oscillations manifest in local field potentials (LFP) in layers II and V of M1 produced by continuous perfusion of kainic acid (100 nM) and carbachol (5 µM). Spontaneous depolarizing GABA-ergic IPSPs in layer V cells, intracellularly dialyzed with KCl and IEM1460 (to block glutamatergic EPSCs), were recorded at -80 mV. IPSPs showed a highly significant (P< 0.01) beta frequency component, which was highly significantly coherent with both the Layer II and V LFP oscillation (which were in antiphase to each other). Both IPSPs and the LFP beta oscillations were abolished by the GABAA antagonist bicuculline. Layer V cells at rest fired spontaneous action potentials at sub-beta frequencies (mean of 7.1+1.2 Hz; n = 27) which were phase-locked to the layer V LFP beta oscillation, preceding the peak of the LFP beta oscillation by some 20 ms. We propose that M1 beta oscillations, in common with other oscillations in other brain regions, can arise from synchronous hyperpolarization of pyramidal cells driven by synaptic inputs from a GABA-ergic interneuronal network (or networks) entrained by recurrent excitation derived from pyramidal cells. This mechanism plays an important role in both the physiology and pathophysiology of control of voluntary movement generation.

An efficient, approximate path-following algorithm for elastic net based nonlinear spike enhancement

Unwanted spike noise in a digital signal is a common problem in digital filtering. However, sometimes the spikes are wanted and other, superimposed, signals are unwanted, and linear, time invariant (LTI) filtering is ineffective because the spikes are wideband - overlapping with independent noise in the frequency domain. So, no LTI filter can separate them, necessitating nonlinear filtering. However, there are applications in which the noise includes drift or smooth signals for which LTI filters are ideal. We describe a nonlinear filter formulated as the solution to an elastic net regularization problem, which attenuates band-limited signals and independent noise, while enhancing superimposed spikes. Making use of known analytic solutions a novel, approximate path-following algorithm is given that provides a good, filtered output with reduced computational effort by comparison to standard convex optimization methods. Accurate performance is shown on real, noisy electrophysiological recordings of neural spikes.