Localization of Subcellular Structures with Super Resolution Light Microscopy on Thin Sections of Nervous Tissue

Release of synaptic vesicles (SV) is a process that is orchestrated by proteins present in the presynaptic terminus called the active zone (AZ). Knowledge of the placement of proteins is necessary to understand how SV release occurs. There is limited information on the location of the AZ proteins from studies of biochemical assays or immuno-electron microscope. Developments in fluorescence light microscopy are capable of reaching subnanometer resolution and therefore can be used to image multiple proteins of the AZ. For instance, a technique called direct Stochastic Optical Reconstruction Microscopy (dSTORM) can reach a resolution of 20 nm in the x-y plane, which is an order of magnitude greater than conventional light microscope. This work is devoted to developing techniques, which enables the use of dSTORM on thick brain tissue samples. In this respect, two thick tissue handling techniques have been explored, namely tomoSTORM and Tokuyasu’s ultracryotomy. Using tomoSTORM, we could construct a super-resolution 3D structure of the calyx of Held synapse. In addition, we also demonstrate multicolor capability by being able to localize the abundantly distributed mitochondria to the synaptic compartment of the calyx of Held. Due to antibody staining limitations, Tokuyasu’s ultracryotomy was explored. Using this approach we gathered dual-color super-resolution data in the calyx of Held on the distribution of Bassoon with respect to Piccolo. In agreement with the standardresolution microscopy, overview image of Bassoon and Piccolo show that both proteins exist together in the majority of the AZs. In addition we can show at the nanoscopic level in a given AZ that the two proteins not only exist as separate entities but are also found to be colocalized. We also gathered data on the distribution of Septin 5 and Piccolo and found that at P7 Septin 5 and Piccolo colocalize while at P17 they do not colocalize. This observation is consistent with the finding that Septin 5 may cluster voltage gated calcium channels at P7 at the AZ. In addition, as dSTORM is limited to photoswitching of 2 dyes, efforts were made to extend this. To this extent, we show efficient photoswitching of phalloidin conjugated to ATTO 488, TRITC and BODIPY 650. 8 In summary, this thesis is focused on adapting dSTORM to thick tissue samples and developing multicolor photoswitching probes to explore multiple protein distribution in the synaptic compartments of mammalian brain tissue

Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy

Determination of the spatial resolution.

<p>(A) point-spread function of a single fluorophore, (B) localization pattern of one single fluorophore that was localized multiple times through reversible photoswitching, (C) histogram of the standard deviation of localizations of 66 single-molecule point-spread functions (average standard deviation 12 nm) and (D) histogram of the full-width half-maximum (FWHM) of 66 single-molecule point-spread functions (average FWHM 28 nm).</p

Dual color <i>d</i>STORM images of mitochondria and their localization within the calyx of Held.

<p>(A) Anti-cytochrome c oxidase stain. Nucleus spared. The soma of the principal neuron is densely populated with mitochodria. (B) Anti-mGFP stain of a single section through the calyx of Held. (C) Overlay of (A) and (B). (D) 3D rendering of a mitochondrion (blue) and surrounding membrane (yellow). Scale bars are 250 nm.</p

Schematic drawing of the experimental approach (see main text for further details).

<p>Schematic drawing of the experimental approach (see main text for further details).</p

Glutaredoxin1 Diminishes Amyloid Beta-Mediated Oxidation of F-Actin and Reverses Cognitive Deficits in an Alzheimer's Disease Mouse Model

Aims: Reactive oxygen species (ROS) generated during Alzheimer's disease (AD) pathogenesis through multiple sources are implicated in synaptic pathology observed in the disease. We have previously shown F-actin disassembly in dendritic spines in early AD (34). The actin cytoskeleton can be oxidatively modified resulting in altered F-actin dynamics. Therefore, we investigated whether disruption of redox signaling could contribute to actin network disassembly and downstream effects in the amyloid precursor protein/presenilin-1 double transgenic (APP/PS1) mouse model of AD. Results: Synaptosomal preparations from 1-month-old APP/PS1 mice showed an increase in ROS levels, coupled with a decrease in the reduced form of F-actin and increase in glutathionylated synaptosomal actin. Furthermore, synaptic glutaredoxin 1 (Grx1) and thioredoxin levels were found to be lowered. Overexpressing Grx1 in the brains of these mice not only reversed F-actin loss seen in APP/PS1 mice but also restored memory recall after contextual fear conditioning. F-actin levels and F-actin nanoarchitecture in spines were also stabilized by Grx1 overexpression in APP/PS1 primary cortical neurons, indicating that glutathionylation of F-actin is a critical event in early pathogenesis of AD, which leads to spine loss. Innovation: Loss of thiol/disulfide oxidoreductases in the synapse along with increase in ROS can render F-actin nanoarchitecture susceptible to oxidative modifications in AD. Conclusions: Our findings provide novel evidence that altered redox signaling in the form of S-glutathionylation and reduced Grx1 levels can lead to synaptic dysfunction during AD pathogenesis by directly disrupting the F-actin nanoarchitecture in spines. Increasing Grx1 levels is a potential target for novel disease-modifying therapies for AD

A beta mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease

Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD(APPswe/PS1 Delta E9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1 Delta E9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1 Delta E9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, A beta load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression