Ilaria Scambi

Exosome derived from murine adipose-derived stromal cells: Neuroprotective effect on in vitro model of amyotrophic lateral sclerosis

Therapeutic strategies for the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) have not yet provided satisfactory results. Interest in stem cells for the treatment of neurodegenerative diseases is increasing and their beneficial action seems to be due to a paracrine effect via the release of exosomes, main mediators of cell-cell communication. Here we wished to assess, in vitro, the efficacy of a novel non-cell therapeutic approach based on the use of exosomes derived from murine adipose-derived stromal cells on motoneuron-like NSC-34 cells expressing ALS mutations, and used as in vitro models of disease. In particular, we set out to investigate the effect of exosomes on NSC-34 naïve cells and NSC-34 cells overexpressing human SOD1(G93A) or SOD1(G37R) or SOD1(A4V) mutants, exposed to oxidative stress. The data presented here indicate for the first time that exosomes (0.2 µg/ml) are able to protect NSC-34 cells from oxidative damage, which is one of the main mechanism of damage in ALS, increasing cell viability. These data highlight a promising role of exosomes derived from stem cells for potential therapeutic applications in motoneuron disease.

Acyl Homoserine Lactones Induce Early Response in the Airway

Acyl homoserine lactones (AHLs) are intercellular signaling molecules used in quorum sensing by Gram‐negative bacteria. We studied the early effects on the rat airway of in vivo intratracheal administration of AHLs (i.e., P. aeruginosa and B. cepacia) to test the hypothesis that AHLs also act on the airway cells, modifying secretory mechanisms which are important in mucosal defense. One hour after treatment, N‐butyryl‐homoserine lactone (C4‐HL) had caused dilated extracellular spaces, loss of cilia, reduction of secretory material, and the presence of prenecrotic elements in the epithelium, while N‐octanoyl‐homoserine lactone (C8‐HL) caused a mild lesion in the epithelium. After treatment with either C4‐ or C8‐HL, reduced immunoreactivity was found using CC10 antibody. At ultrastructural examination, dilatation of the mitochondria was evident in ciliate and secretory cells, while solitary chemosensory cells appeared better preserved, showing aspects of nucleocytoplasmic activation. Using microarray analysis, we found down‐regulation of early gene Fos and Egr1 in all AHL‐treated specimens. In vivo pharmacological magnetic resonance imaging after C4‐ or C8‐HL treatment showed a slight increase in tracheal secretion at a first evaluation 5 min after administration, with no increase in the following minutes. In conclusion, AHLs induce an early mucosal response, and the chondriomas of ciliate and secretory cells are the main cytological target of AHL action. Our results show that AHL action is not limited to activation of conspecific bacteria, but also modifies innate airway defense mechanisms. Anat Rec, 292:439–448, 2009.

Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes

Purpose Recent findings indicate that the beneficial effects of adipose stem cells (ASCs), reported in several neurodegenerative experimental models, could be due to their paracrine activity mediated by the release of exosomes. The aim of this study was the development and validation of an innovative exosome-labeling protocol that allows to visualize them with magnetic resonance imaging (MRI). Materials and methods At first, ASCs were labeled using ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4–6 nm), and optimal parameters to label ASCs in terms of cell viability, labeling efficiency, iron content, and magnetic resonance (MR) image contrast were investigated. Exosomes were then isolated from labeled ASCs using a standard isolation protocol. The efficiency of exosome labeling was assessed by acquiring MR images in vitro and in vivo as well as by determining their iron content. Transmission electron microscopy images and histological analysis were performed to validate the results obtained. Results By using optimized experimental parameters for ASC labeling (200 µg Fe/mL of USPIO and 72 hours of incubation), it was possible to label 100% of the cells, while their viability remained comparable to unlabeled cells; the detection limit of MR images was of 102 and 2.5×103 ASCs in vitro and in vivo, respectively. Exosomes isolated from previously labeled ASCs retain nanoparticles, as demonstrated by transmission electron microscopy images. The detection limit by MRI was 3 µg and 5 µg of exosomes in vitro and in vivo, respectively. Conclusion We report a new approach for labeling of exosomes by USPIO that allows detection by MRI while preserving their morphology and physiological characteristics.

Increased anxiety-like behavior and selective learning impairments are concomitant to loss of hippocampal interneurons in the presymptomatic SOD1(G93A) ALS mouse model.

Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease primarily characterized by motor neuron death, causes damages beyond motor‐related areas. In particular, cognitive impairments and hippocampal damage have been reported in ALS patients. We investigated spatial navigation learning and hippocampal interneurons in a mutant SOD1(G93A) mouse (mSOD1) model of ALS. Behavioral tests were performed by using presymptomatic mSOD1 mice. General motor activity was comparable to that of wild‐type mice in the open‐field test, in which, however mSOD1 exhibited increased anxiety‐like behavior. In the Barnes maze test, mSOD1 mice displayed a delay in learning, outperformed wild‐type mice during the first probe trial, and exhibited impaired long‐term memory. Stereological counts of parvalbumin‐positive interneurons, which are crucial for hippocampal physiology and known to be altered in other central nervous system regions of mSOD1 mice, were also performed. At postnatal day (P) 56, the population of parvalbumin‐positive interneurons in mSOD1 mice was already reduced in CA1 and in CA3, and at P90 the reduction extended to the dentate gyrus. Loss of parvalbumin‐positive hippocampal interneurons occurred mostly during the presymptomatic stage. Western blot analysis showed that hippocampal parvalbumin expression levels were already reduced in mSOD1 mice at P56. The hippocampal alterations in mSOD1 mice could at least partly account for the increased anxiety‐like behavior and deficits in spatial navigation learning. Our study provides evidence for cognitive alterations and damage to the γ‐aminobutyric acid (GABA)ergic system in the hippocampus of murine ALS, thereby revealing selective deficits antecedent to the onset of motor symptoms. J. Comp. Neurol. 523:1622–1638, 2015.

A microRNA signature from serum exosomes of patients with glioma as complementary diagnostic biomarker

Malignant gliomas, the most frequent primary brain tumors, are characterized by a dismal prognosis. Reliable biomarkers complementary to neuroradiology in the differential diagnosis of gliomas and monitoring for post-surgical progression are unmet needs. Altered expression of several microRNAs in tumour tissues from patients with gliomas compared to normal brain tissue have been described, thus supporting the rationale of using microRNA-based biomarkers. Although different circulating microRNAs were proposed in association with gliomas, they have not been introduced into clinical practice so far. Blood samples were collected from patients with high and low grade gliomas, both before and after surgical resection, and the expression of miR-21, miR-222 and miR-124-3p was measured in exosomes isolated from serum. The expression levels of miR-21, miR-222 and miR-124-3p in serum exosomes of patients with high grade gliomas were significantly higher than those of low grade gliomas and healthy controls and were sharply decreased in samples obtained after surgery. The analysis of miR-21, miR-222 and miR-124-3p in serum exosomes of patients affected by gliomas can provide a minimally invasive and innovative tool to help the differential diagnosis of gliomas at their onset in the brain and predict glioma grading and non glial metastases before surgery.

Labeling and Magnetic Resonance Imaging of Exosomes Isolated from Adipose Stem Cells

Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation‐free operation. To be detectable with MRI, exosomes must be labeled with MR contrast agents, such as ultra‐small superparamagnetic iron oxide nanoparticles (USPIO). Here, we set up an innovative approach for exosome labeling that preserves their morphology and physiological characteristics. We show that by labeling ASC with USPIO before extraction of nanovesicles, the isolated exosomes retain nanoparticles and can be visualized by MRI. The current work aims at validating this novel USPIO‐based exosome labeling method by monitoring the efficiency of the labeling with MRI both in ASC and in exosomes.

MRI reveals therapeutical efficacy of stem cells: An experimental study on the SOD1(G93A) animal model

The first part of the experiment identifies and validates MRI biomarkers distinctive of the disease progression in the transgenic superoxide dismutase gene (SOD1(G93A)) animal model. The second part assesses the efficacy of a mesenchymal stem cell‐based therapy through the MRI biomarkers previously defined.

Acetylation state of RelA modulated by epigenetic drugs prolongs survival and induces a neuroprotective effect on ALS murine model

Dysregulation in acetylation homeostasis has been implicated in the pathogenesis of the amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. It is known that the acetylation of transcriptional factors regulates their activity. The acetylation state of NF-kB RelA has been found to dictate the neuroprotective versus the neurotoxic effect of p50/RelA. Here we showed that the pro-apoptotic acetylation mode of RelA, involving a general lysine deacetylation of the subunit with the exclusion of the lysine 310, is evident in the lumbar spinal cord of SOD1(G93A) mice, a murine model of ALS. The administration of the HDAC inhibitor MS-275 and the AMPK/sirtuin 1 activator resveratrol restored the normal RelA acetylation in SOD1(G93A) mice. The SOD1(G93A) mice displayed a 3 weeks delay of the disease onset, associated with improvement of motor performance, and 2 weeks increase of lifespan. The epigenetic treatment rescued the lumbar motor neurons affected in SOD1(G93A) mice, accompanied by increased levels of protein products of NF-kB-target genes, Bcl-xL and brain-derived neurotrophic factor. In conclusion, we here demonstrate that MS-275 and resveratrol restore the acetylation state of RelA in the spinal cord, delaying the onset and increasing the lifespan of SOD1(G93A) mice.