Measuring mRNA translation in neuronal processes and somata by tRNA-FRET.

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

Aß 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/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 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/PS1ΔE9 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, Aß load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression.SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.

Characterization of Nanoscale Organization of F-Actin in Morphologically Distinct Dendritic Spines In Vitro Using Supervised Learning.

The cytoarchitecture of a neuron is very important in defining morphology and ultrastructure. Although there is a wealth of information on the molecular components that make and regulate these ultrastructures, there is a dearth of understanding of how these changes occur or how they affect neurons in health and disease. Recent advances in nanoscale imaging which resolve cellular structures at the scale of tens of nanometers below the limit of diffraction enable us to understand these structures in fine detail. However, automated analysis of these images is still in its infancy. Towards this goal, attempts have been made to automate the detection and analysis of the cytoskeletal organization of microtubules. To date, evaluation of the nanoscale organization of filamentous actin (F-actin) in neuronal compartments remains challenging. Here, we present an objective paradigm for analysis which adopts supervised learning of nanoscale images of F-actin network in excitatory synapses, obtained by single molecule based super-resolution light microscopy. We have used the proposed analysis to understand the heterogeneity in the organization of F-actin in dendritic spines of primary neuronal cultures from rodents. Our results were validated using ultrastructural data obtained from platinum replica electron microscopy (PREM). The automated analysis approach was used to differentiate the heterogeneity in the nanoscale organization of F-actin in primary neuronal cultures from wild-type (WT) and a transgenic mouse model of Alzheimer's disease (APPSwe/PS1ΔE9).

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.

Single-molecule super-resolution imaging by tryptophan-quenching-induced photoswitching of phalloidin-fluorophore conjugates.

Photophysical properties of any fluorophore are governed by the chemical nanoenvironment. In the context of imaging biological samples, this translates to different photophysical properties for different labels and probes. Here, we demonstrate that the nanoenvironment of fluorophores within a probe can be advantageously used to induce particular properties such as light-induced photoswitching. We demonstrate efficient photoswitching and single-molecule super-resolution imaging for various fluorophore-phalloidin conjugates in aqueous buffer without the addition of further chemicals. We further demonstrate the utility of two-color imaging of fluorophore-phalloidin and a photoactivatable fluorescent protein in presynaptic nerve terminals.

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.

Anti-androgenic endocrine disrupting activities of chlorpyrifos and piperophos.

The present work describes the screening and characterization of some common endocrine disrupting chemicals for their (anti)androgenic activities. Various chemicals (mostly pesticides and pharmaceuticals) were screened with the NIH3T3 cell line stably expressing human androgen receptor (hAR) and luciferase reporter gene for their ability to stimulate luciferase activity or inhibit the response that was evoked by 0.4nM testosterone. The most potent anti-androgenic compounds identified in our assay included chlorpyrifos, endosulfan and piperophos. Finally, the chemicals were analyzed for their effects on steriodogenesis in rat Leydig cells. Piperophos and chlorpyrifos showed a significant decrease in testosterone biosynthesis by Leydig cells. RT-PCR studies showed decrease in the expression of key steroidogenic enzymes: cytochrome P450scc, 3beta-HSD and 17beta-HSD and immunoblot analysis demonstrated a decrease in steroidogenic acute regulatory (StAR) protein expression by both these chemicals. Chlorpyrifos also showed a decrease in LH receptor stimulated cAMP production. In conclusion, we demonstrate that commonly used pesticides like chlorpyrifos and piperophos pose serious threat to male reproductive system by interfering at various levels of androgen biosynthesis.