Release of VAMP5-positive extracellular vesicles by retinal Müller glia in vivo.

Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles. The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of radial glial cells in the retina, which forms part of the central nervous system. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct extracellular vesicles from endfeet and microvilli of retinal Müller cells in adult mice in vivo. We identify VAMP5 as a Müller cell-specific SNARE component that is part of extracellular vesicles and responsive to ischemia, and we reveal differences between the secretomes of immunoaffinity-purified Müller cells and neurons in vitro. Our findings suggest extracellular vesicle-based communication as an important mediator of cellular interactions in the retina.

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neurons in vivo.

Niemann-Pick type C disease is a rare and fatal lysosomal storage disorder presenting severe neurovisceral symptoms. Disease-causing mutations in genes encoding either NPC1 or NPC2 protein provoke accumulation of cholesterol and other lipids in specific structures of the endosomal-lysosomal system and degeneration of specific cells, notably neurons in the central nervous system (CNS). 2-hydroxypropyl-beta-cyclodextrin (CD) emerged as potential therapeutic approach based on animal studies and clinical data, but the mechanism of action in neurons has remained unclear. To address this topic in vivo, we took advantage of the retina as highly accessible part of the CNS and intravitreal injections as mode of drug administration. Coupling CD to gold nanoparticles allowed us to trace its intracellular location. We report that CD enters the endosomal-lysosomal system of neurons in vivo and enables the release of lipid-laden lamellar inclusions, which are then removed from the extracellular space by specific types of glial cells. Our data suggest that CD induces a concerted action of neurons and glial cells to restore lipid homeostasis in the central nervous system.

Glial markers and emotional memory in rats following acute cerebral radiofrequency exposures.

The widespread mobile phone use raises concerns on the possible cerebral effects of radiofrequency electromagnetic fields (RF EMF). Reactive astrogliosis was reported in neuroanatomical structures of adaptive behaviors after a single RF EMF exposure at high specific absorption rate (SAR, 6 W/kg). Here, we aimed to assess if neuronal injury and functional impairments were related to high SAR-induced astrogliosis. In addition, the level of beta amyloid 1-40 (Aß 1-40) peptide was explored as a possible toxicity marker. Sprague Dawley male rats were exposed for 15 min at 0, 1.5, or 6 W/kg or for 45 min at 6 W/kg. Memory, emotionality, and locomotion were tested in the fear conditioning, the elevated plus maze, and the open field. Glial fibrillary acidic protein (GFAP, total and cytosolic fractions), myelin basic protein (MBP), and Aß1-40 were quantified in six brain areas using enzyme-linked immunosorbent assay. According to our data, total GFAP was increased in the striatum (+114 %) at 1.5 W/kg. Long-term memory was reduced, and cytosolic GFAP was increased in the hippocampus (+119 %) and in the olfactory bulb (+46 %) at 6 W/kg (15 min). No MBP or Aß1-40 expression modification was shown. Our data corroborates previous studies indicating RF EMF-induced astrogliosis. This study suggests that RF EMF-induced astrogliosis had functional consequences on memory but did not demonstrate that it was secondary to neuronal damage.

Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions.

UNLABELLED: Aging and pathologic conditions cause intracellular aggregation of macromolecules and the dysfunction and degeneration of neurons, but the mechanisms are largely unknown. Prime examples are lysosomal storage disorders such as Niemann-Pick type C (NPC) disease, where defects in the endosomal-lysosomal protein NPC1 or NPC2 cause intracellular accumulation of unesterified cholesterol and other lipids leading to neurodegeneration and fatal neurovisceral symptoms. Here, we investigated the impact of NPC1 deficiency on rodent neurons using pharmacologic and genetic models of the disease. Improved ultrastructural detection of lipids and correlative light and electron microscopy identified lamellar inclusions as the subcellular site of cholesterol accumulation in neurons with impaired NPC1 activity. Immunogold labeling combined with transmission electron microscopy revealed the presence of CD63 on internal lamellae and of LAMP1 on the membrane surrounding the inclusions, indicating their origins from intraluminal vesicles of late endosomes and of a lysosomal compartment, respectively. Lamellar inclusions contained cell-intrinsic cholesterol and surface-labeled GM1, indicating the incorporation of plasma membrane components. Scanning electron microscopy revealed that the therapeutic drug candidate ß-cyclodextrin induces the subplasmalemmal location of lamellar inclusions and their subsequent release to the extracellular space. In parallel, ß-cyclodextrin mediated the NPC1-independent redistribution of cholesterol within neurons and thereby abolished a deleterious cycle of enhanced cholesterol synthesis and its intracellular accumulation, which was indicated by neuron-specific transcript analysis. Our study provides new mechanistic insight into the pathologic aggregation of macromolecules in neurons and suggests exocytosis as cellular target for its therapeutic reversal. SIGNIFICANCE STATEMENT: Many neurodegenerative diseases involve pathologic accumulation of molecules within neurons, but the subcellular location and the cellular impact are often unknown and therapeutic approaches lacking. We investigated these questions in the lysosomal storage disorder Niemann-Pick type C (NPC), where a defect in intracellular cholesterol transport causes loss of neurons and fatal neurovisceral symptoms. Here, we identify lamellar inclusions as the subcellular site of lipid accumulation in neurons, we uncover a vicious cycle of cholesterol synthesis and accretion, which may cause gradual neurodegeneration, and we reveal how ß-cyclodextrin, a potential therapeutic drug, reverts these changes. Our study provides new mechanistic insight in NPC disease and uncovers new targets for therapeutic approaches.

Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer.

PURPOSE: The diagnostic test for ALK rearrangement in non-small-cell lung cancer (NSCLC) for crizotinib treatment is currently done on tumor biopsies or fine-needle aspirations. We evaluated whether ALK rearrangement diagnosis could be performed by using circulating tumor cells (CTCs). PATIENTS AND METHODS: The presence of an ALK rearrangement was examined in CTCs of 18 ALK-positive and 14 ALK-negative patients by using a filtration enrichment technique and filter-adapted fluorescent in situ hybridization (FA-FISH), a FISH method optimized for filters. ALK-rearrangement patterns were determined in CTCs and compared with those present in tumor biopsies. ALK-rearranged CTCs and tumor specimens were characterized for epithelial (cytokeratins, E-cadherin) and mesenchymal (vimentin, N-cadherin) marker expression. ALK-rearranged CTCs were monitored in five patients treated with crizotinib. RESULTS: All ALK-positive patients had four or more ALK-rearranged CTCs per 1 mL of blood (median, nine CTCs per 1 mL; range, four to 34 CTCs per 1 mL). No or only one ALK-rearranged CTC (median, one per 1 mL; range, zero to one per 1 mL) was detected in ALK-negative patients. ALK-rearranged CTCs harbored a unique (3'5') split pattern, and heterogeneous patterns (3'5', only 3') of splits were present in tumors. ALK-rearranged CTCs expressed a mesenchymal phenotype contrasting with heterogeneous epithelial and mesenchymal marker expressions in tumors. Variations in ALK-rearranged CTC levels were detected in patients being treated with crizotinib. CONCLUSION: ALK rearrangement can be detected in CTCs of patients with ALK-positive NSCLC by using a filtration technique and FA-FISH, enabling both diagnostic testing and monitoring of crizotinib treatment. Our results suggest that CTCs harboring a unique ALK rearrangement and mesenchymal phenotype may arise from clonal selection of tumor cells that have acquired the potential to drive metastatic progression of ALK-positive NSCLC.

Circulating tumor cells in lung cancer.

Circulating tumor cells (CTCs) have emerged as potential biomarkers in several cancers such as colon, prostate, and breast carcinomas, with a correlation between CTC number and patient prognosis being established by independent research groups. The detection and enumeration of CTCs, however, is still a developing field, with no universal method of detection suitable for all types of cancer. CTC detection in lung cancer in particular has proven difficult to perform, as CTCs in this type of cancer often present with nonepithelial characteristics. Moreover, as many detection methods rely on the use of epithelial markers to identify CTCs, the loss of these markers during epithelial-to-mesenchymal transition in certain metastatic cancers can render these methods ineffective. The development of personalized medicine has led to an increase in the advancement of molecular characterization of CTCs. The application of techniques such as FISH and RT-PCR to detect EGFR, HER2, and KRAS abnormalities in lung, breast, and colon cancer, for example, could be used to characterize CTCs in real time. The use of CTCs as a 'liquid biopsy' is therefore an exciting possibility providing information on patient prognosis and treatment efficacy. This review summarizes the state of CTC detection today, with particular emphasis on lung cancer, and discusses the future applications of CTCs in helping the clinician to develop new strategies in patient treatment.