Person:

Breakefield, Xandra

We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared fluorescent proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted fluorescent proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.

Background: RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients. Methods: Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups). Results: Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production. Conclusions: Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.

Extracellular vesicles (microvesicles), such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. Microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. Here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. Herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. In addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. We highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. Finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. More research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies.

Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3′-untranslated region (3′UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3′UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs.

Most cases of early onset DYT1 dystonia in humans are caused by a GAG deletion in the TOR1A gene leading to loss of a glutamic acid ((\Delta)E) in the torsinA protein, which underlies a movement disorder associated with neuronal dysfunction without apparent neurodegeneration. Mutation/deletion of the gene (Dst) encoding dystonin in mice results in a dystonic movement disorder termed dystonia musculorum, which resembles aspects of dystonia in humans. While torsinA and dystonin proteins do not share modular domain architecture, they participate in a similar function by modulating a structural link between the nuclear envelope and the cytoskeleton in neuronal cells. We suggest that through a shared interaction with the nuclear envelope protein nesprin-3(\alpha), torsinA and the neuronal dystonin-a2 isoform comprise a bridge complex between the outer nuclear membrane and the cytoskeleton, which is critical for some aspects of neuronal development and function. Elucidation of the overlapping roles of torsinA and dystonin-a2 in nuclear/endoplasmic reticulum dynamics should provide insights into the cellular mechanisms underlying the dystonic phenotype.

Extracellular membrane vesicles (EMVs) are nanometer sized vesicles, including exosomes and microvesicles capable of transferring DNAs, mRNAs, microRNAs, non-coding RNAs, proteins, and lipids among cells without direct cell-to-cell contact, thereby representing a novel form of intercellular communication. Many cells in the nervous system have been shown to release EMVs, implicating their active roles in development, function, and pathologies of this system. While substantial progress has been made in understanding the biogenesis, biophysical properties, and involvement of EMVs in diseases, relatively less information is known about their biological function in the normal nervous system. In addition, since EMVs are endogenous vehicles with low immunogenicity, they have also been actively investigated for the delivery of therapeutic genes/molecules in treatment of cancer and neurological diseases. The present review summarizes current knowledge about EMV functions in the nervous system under both physiological and pathological conditions, as well as emerging EMV-based therapies that could be applied to the nervous system in the foreseeable future.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

TorsinA is an (AAA^+) ATPase located within the lumen of the endoplasmic reticulum and nuclear envelope, with a mutant form causing early onset torsion dystonia (DYT1). Here we report a new function for torsinA in endoplasmic reticulum-associated degradation (ERAD). Retro-translocation and proteosomal degradation of a mutant cystic fibrosis transmembrane conductance regulator ((CFTR\Delta F508)) was inhibited by downregulation of torsinA or overexpression of mutant torsinA, and facilitated by increased torsinA. Retro-translocation of cholera toxin was also decreased by downregulation of torsinA. TorsinA associates with proteins implicated in ERAD, including Derlin-1, VIMP, and p97. Further, torsinA reduces endoplasmic reticulum stress in nematodes overexpressing (CFTR\Delta F508), and fibroblasts from DYT1 dystonia patients are more sensitive than controls to endoplasmic reticulum stress and less able to degrade mutant CFTR. Therefore, compromised ERAD function in the cells of DYT1 patients may increase sensitivity to endoplasmic reticulum stress with consequent alterations in neuronal function contributing to the disease state.

Background: The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). Methodology/Principle Findings: An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. Conclusions/Significance: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.