Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase.

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a "naked" state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope.

Direct imaging of uncoated biological samples enables correlation of super-resolution and electron microscopy data.

A simple method for imaging biological tissue samples by electron microscopy and its correlation with super-resolution light microscopy is presented. This room temperature protocol, based on protecting thin biological specimens with methylcellulose and imaging with low voltage scanning electron microscopy, circumvents complex classical electron microscopy sample preparation steps requiring dehydration, resin embedding and use of contrast agents. This technique facilitates visualization of subcellular structures e.g. synaptic clefts and synaptic vesicles in mouse brain tissue and the organization of mitochondrial cristae in the zebrafish retina. Application of immunogold protocols to these samples can determine the precise localization of synaptic proteins and, in combination with super-resolution light microscopy methods clearly pinpoints the subcellular distribution of several proteins in the tissue. The simplicity of the method, including section collection on a silicon wafer, reduces artefacts and correlates protein location with sample morphology.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy.

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue.

Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent form of age-related dementia, and its effect on society increases exponentially as the population ages. Accumulating evidence suggests that neuroinflammation, mediated by the brain's innate immune system, contributes to AD neuropathology and exacerbates the course of the disease. However, there is no experimental evidence for a causal link between systemic inflammation or neuroinflammation and the onset of the disease. METHODS: The viral mimic, polyriboinosinic-polyribocytidilic acid (PolyI:C) was used to stimulate the immune system of experimental animals. Wild-type (WT) and transgenic mice were exposed to this cytokine inducer prenatally (gestation day (GD)17) and/or in adulthood. Behavioral, immunological, immunohistochemical, and biochemical analyses of AD-associated neuropathologic changes were performed during aging. RESULTS: We found that a systemic immune challenge during late gestation predisposes WT mice to develop AD-like neuropathology during the course of aging. They display chronic elevation of inflammatory cytokines, an increase in the levels of hippocampal amyloid precursor protein (APP) and its proteolytic fragments, altered Tau phosphorylation, and mis-sorting to somatodendritic compartments, and significant impairments in working memory in old age. If this prenatal infection is followed by a second immune challenge in adulthood, the phenotype is strongly exacerbated, and mimics AD-like neuropathologic changes. These include deposition of APP and its proteolytic fragments, along with Tau aggregation, microglia activation and reactive gliosis. Whereas Aß peptides were not significantly enriched in extracellular deposits of double immune-challenged WT mice at 15 months, they dramatically increased in age-matched immune-challenged transgenic AD mice, precisely around the inflammation-induced accumulations of APP and its proteolytic fragments, in striking similarity to the post-mortem findings in human patients with AD. CONCLUSION: Chronic inflammatory conditions induce age-associated development of an AD-like phenotype in WT mice, including the induction of APP accumulations, which represent a seed for deposition of aggregation-prone peptides. The PolyI:C mouse model therefore provides a unique tool to investigate the molecular mechanisms underlying the earliest pathophysiological changes preceding fibrillary Aß plaque deposition and neurofibrillary tangle formations in a physiological context of aging. Based on the similarity between the changes in immune-challenged mice and the development of AD in humans, we suggest that systemic infections represent a major risk factor for the development of AD.

Extrusion of misfolded and aggregated proteins--a protective strategy of aging neurons?

Cellular senescence is the consequence of repetitive exposures to oxidative stress, perturbed energy homeostasis, accumulation of damaged proteins and lesions in their nucleic acids. Whereas mitotic cells are equipped with efficient cell replacement strategies; postmitotic neurons have--with a few exceptions--no mechanism to substitute dysfunctional cells within a complex neuronal network. Here we propose a potential strategy by which aging neurons contend against abnormal accumulation of damaged/misfolded proteins. The suggested mechanism involves the formation of 'budding-like' extrusions and their subsequent clearance by glia. This hypothesis emerged from our previous investigations of the aged hippocampus revealing layer-specific accumulations of Reelin, a glycoprotein with fundamental roles during brain development and adult synaptic plasticity. We showed that Reelin deposits constitute a conserved neuropathological feature of aging, which is significantly accelerated in adult wild-type mice prenatally exposed to a viral-like infection. Here, we employed two- and three-dimensional immunoelectron microscopy to elucidate their morphological properties, localization and origin in immune challenged vs. control mice. In controls, Reelin-positive deposits were dispersed in the neuropil, some being engulfed by glia. In immune challenged mice, however, significantly more Reelin-immunoreactive deposits were associated with neuritic swellings containing mitochondria, vacuoles and cellular debris, pointing to their intracellular origin and suggesting that 'budding-like' neuronal extrusions of misfolded proteins and glial clearance may represent a protective strategy to counteract aging-associated impairments in proteosomal/lysosomal degradation. Neurons exposed to chronic neuroinflammation with increased levels of misfolded/damaged proteins, however, may fail to combat intraneuronal protein accumulations, a process probably underlying neuronal dysfunction and degeneration during aging.

GABA neurons of the VTA drive conditioned place aversion.

Salient but aversive stimuli inhibit the majority of dopamine (DA) neurons in the ventral tegmental area (VTA) and cause conditioned place aversion (CPA). The cellular mechanism underlying DA neuron inhibition has not been investigated and the causal link to behavior remains elusive. Here, we show that GABA neurons of the VTA inhibit DA neurons through neurotransmission at GABA(A) receptors. We also observe that GABA neurons increase their firing in response to a footshock and provide evidence that driving GABA neurons with optogenetic effectors is sufficient to affect behavior. Taken together, our data demonstrate that synaptic inhibition of DA neurons drives place aversion.

Reduced Reelin expression accelerates amyloid-beta plaque formation and tau pathology in transgenic Alzheimer's disease mice.

In addition to the fundamental role of the extracellular glycoprotein Reelin in neuronal development and adult synaptic plasticity, alterations in Reelin-mediated signaling have been suggested to contribute to neuronal dysfunction associated with Alzheimer's disease (AD). In vitro data revealed a biochemical link between Reelin-mediated signaling, Tau phosphorylation, and amyloid precursor protein (APP) processing. To directly address the role of Reelin in amyloid-beta plaque and Tau pathology in vivo, we crossed heterozygous Reelin knock-out mice (reeler) with transgenic AD mice to investigate the temporal and spatial AD-like neuropathology. We demonstrate that a reduction in Reelin expression results in enhanced amyloidogenic APP processing, as indicated by the precocious production of amyloid-beta peptides, the significant increase in number and size of amyloid-beta plaques, as well as age-related aggravation of plaque pathology in double mutant compared with single AD mutant mice of both sexes. Numerous amyloid-beta plaques accumulate in the hippocampal formation and neocortex of double mutants, precisely in layers with strongest Reelin expression and highest accumulation of Reelin plaques in aged wild-type mice. Moreover, concentric accumulations of phosphorylated Tau-positive neurons around amyloid-beta plaques were evident in 15-month-old double versus single mutant mice. Silver stainings indicated the presence of neurofibrillary tangles, selectively associated with amyloid-beta plaques and dystrophic neurites in the entorhinal cortex and hippocampus. Our findings suggest that age-related Reelin aggregation and concomitant reduction in Reelin-mediated signaling play a proximal role in synaptic dysfunction associated with amyloid-beta deposition, sufficient to enhance Tau phosphorylation and tangle formation in the hippocampal formation in aged Reelin-deficient transgenic AD mice.

Co-localization of Reelin and proteolytic AbetaPP fragments in hippocampal plaques in aged wild-type mice.

Reelin is a large extracellular glycoprotein required for proper neuronal positioning during development. In the adult brain, Reelin plays a crucial modulatory role in the induction of synaptic plasticity and successful formation of long-term memory. Recently, alterations in Reelin-mediated signaling have been suggested to contribute to neuronal dysfunction associated with Alzheimer's disease (AD). We previously reported that aging in several species is characterized by a decline in Reelin-expressing interneurons and concomitant accumulation in amyloid-like plaques in the hippocampal formation, significantly correlating with cognitive impairments. In transgenic AD mice, we detected Reelin in oligomeric amyloid-beta aggregates and in tight association with fibrillary plaques. Here, we used immunohistochemistry at the light and electron microscopy level to characterize further the morphology, temporal and spatial progression, as well as the potential of Reelin-positive plaques to sequester murine amyloid-beta peptides in wild-type mice. We developed a new immunohistochemical protocol involving a stringent protease pretreatment which markedly enhanced Reelin-immunoreactivity and allowed specific detection of variable shapes of murine anti-amyloid-beta protein precursor-immunoreactivity in plaques in the hippocampus, likely representing N-terminal fragments and amyloid-beta species. Ultrastructural investigations confirmed the presence of Reelin in extracellular space, somata of interneurons in young and aged wild-type mice. In aged mice, Reelin- and amyloid-beta-immunoreactivity was detected in extracellular, spherical deposits, likely representing small intermediates or fragments of amyloid fibrils. Our results suggest that Reelin itself aggregates into abnormal oligomeric or protofibrillary deposits during aging, potentially creating a precursor condition for fibrillary amyloid-beta plaque formation.

Reelin-mediated Signaling during Normal and Pathological Forms of Aging.

Reelin is a large extracellular matrix protein essential for mediating proper neuronal positioning during development. Employing the same lipoprotein-mediated signaling cascade, Reelin regulates NMDA receptor homeostasis and modulates synaptic function and plasticity in adult synapses. In line, aging-related reduction in Reelin expression has been shown to contribute to cognitive impairments during normal aging. Although recent experimental evidence suggests an involvement of dysfunctional Reelin in pathological forms of aging, such as late-onset Alzheimer's disease (AD), the molecular mechanisms by which this conserved extracellular glycoprotein contributes to the pathogenesis of AD remains still largely unknown. In the present review, we briefly summarize the role of Reelin in the developing and adult brain and discuss the implication of loss- or gain-of-functions of developmental programs in the adult brain as putative inducing factors of pathological forms of aging. Finally, we will propose some new concepts on the role of inflammatory cytokines in interfering with Reelin-mediated signaling during neurodevelopment and adult synaptic function, and discuss how this could be translated into a novel non-transgenic mouse model of late-onset AD. Thus, the findings presented in this review are aimed to highlight the important role of Reelin-mediated signaling in maintaining a crucial developmental program in the adult brain that is required to prevent the shift from normal to pathological aging.

Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin.

Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu(9), Glu(194), and Glu(204), were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.

An elevated level of cholesterol impairs self-assembly of pulmonary surfactant into a functional film.

In adult respiratory distress syndrome, the primary function of pulmonary surfactant to strongly reduce the surface tension of the air-alveolar interface is impaired, resulting in diminished lung compliance, a decreased lung volume, and severe hypoxemia. Dysfunction coincides with an increased level of cholesterol in surfactant which on its own or together with other factors causes surfactant failure. In the current study, we investigated by atomic force microscopy and Kelvin-probe force microscopy how the increased level of cholesterol disrupts the assembly of an efficient film. Functional surfactant films underwent a monolayer-bilayer conversion upon contraction and resulted in a film with lipid bilayer stacks, scattered over a lipid monolayer. Large stacks were at positive electrical potential, small stacks at negative potential with respect to the surrounding monolayer areas. Dysfunctional films formed only few stacks. The surface potential of the occasional stacks was also not different from the surrounding monolayer. Based on film topology and potential distribution, we propose a mechanism for formation of stacked bilayer patches whereby the helical surfactant-associated protein SP-C becomes inserted into the bilayers with defined polarity. We discuss the functional role of the stacks as mechanically reinforcing elements and how an elevated level of cholesterol inhibits the formation of the stacks. This offers a simple biophysical explanation for surfactant inhibition in adult respiratory distress syndrome and possible targets for treatment.