Function of exosomes in neurological disorders and brain tumors

Exosomes are a subtype of extracellular vesicles released from different cell types including those in the nervous system, and are enriched in a variety of bioactive molecules such as RNAs, proteins and lipids. Numerous studies have indicated that exosomes play a critical role in many physiological and pathological activities by facilitating intercellular communication and modulating cells’ responses to external environments. Particularly in the central nervous system, exosomes have been implicated to play a role in many neurological disorders such as abnormal neuronal development, neurodegenerative diseases, epilepsy, mental disorders, stroke, brain injury and brain cancer. Since exosomes recapitulate the characteristics of the parental cells and have the capacity to cross the blood-brain barrier, their cargo can serve as potential biomarkers for early diagnosis and clinical assessment of disease treatment. In this review, we describe the latest findings and current knowledge of the roles exosomes play in various neurological disorders and brain cancer, as well as their application as promising biomarkers. The potential use of exosomes to deliver therapeutic molecules to treat diseases of the central nervous system is also discussed

Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12-48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer

Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells

Background: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. Methods: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. Results: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. Conclusions: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications

Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells

BACKGROUND: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. METHODS: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. RESULTS: Exosomes released from cancer cells contain mRNA and protein. mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and , a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. CONCLUSIONS: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications

Pharmacological inhibition of UPR increases self-complementary AAV2 mediated transgene expression in vivo.

<p>C57/BL6 mice were either mock injected or injected with scAAV2 alone or with metformin and scAAV2 vectors at a dose of 1×10¹¹ vgs per mouse. Four weeks later, mice were euthanized and the liver lobes were studied for EGFP expression by fluorescence microscopy. All images were taken at an identical exposure of 576 milliseconds, gain of 1.5 and an intensity of 2. <b>A.</b> Representative images from each of the groups. <b>B.</b> Images from five visual fields per group were analyzed quantitatively using image-J software. *p<0.05 <i>Vs</i> scAAV2 treated mice.</p

Comparative analysis of AAV mediated transduction efficiency in HeLa cells after siRNA mediated knock down of PERK or IRE1α pathways.

<p><b>A.</b> Transgene expression was measured in HeLa cells 48 hrs post-infection with self-complementary AAV2-EGFP or AAV6-EFGP vectors either in the presence or absence of specific siRNA or scrambled siRNA control. <b>B.</b> Quantitative analyses of the data from (A) by fluorescence microscopy. Images from five visual fields were analyzed quantitatively by ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel²) per visual field (mean ± SD) and normalized to 1 for the control. Error bars represent standard error and the graph is a representative data set of at least three independent experiments. *p<0.05 <i>Vs</i> scrambled siRNA treated cells <b>C.</b> Western blot analysis of HeLa cellular extracts following mock (PBS)-infection or infection with AAV vectors, either in the presence or absence of PERK or IRE1α siRNA or scrambled siRNA control. β-actin was used as a loading control.</p

Alternate serotypes AAV 1 and AAV6 induce cellular unfolded protein response.

<p><b>A.</b> HeLa cells were infected with 5,000 vgs/cell of scAAV1- EGFP or scAAV6-EGFP vectors under identical conditions. Twelve hours post infection, the differential gene expression of UPR targets were assessed between mock-infected or AAV infected cells. Expression level of PERK, ATF6, IRE1 and CHOP from cells treated with AAV1 and AAV6. DTT (Dithiothreitol) was used as a positive control of UPR activation. *p<0.05 <i>Vs</i> mock infected cells.</p

Self-complementary AAV2 infection activates PERK1 and IRE1α pathway and its downstream targets.

<p><b>A.</b> Total RNA from HeLa cells mock-infected or infected with of 5,000 vgs/cell of scAAV2-CB-EGFP vectors was used to profile the expression of downstream targets of IRE1α and PERK target genes such as ATF4 or CHOP by real-time PCR analysis at 2, 6, 12, 24 and 48 hours post infection. *p<0.05 <i>Vs</i> mock infected cells. <b>B.</b> Qualitative reverse-transcription PCR amplification of XBP1 (283 bp) and spliced variant sXBP1 (257 bp) at various time points, 2 h (lane 1), 6 h (lane 2), Molecular weight ladder (lane 3), 12 h (lane 4), 24 h (lane 5) and 48 h (lane 6) analyzed. Dithiothreitol (DTT, lane 7) was used as a positive control of UPR activation.</p

Comparative gene expression profiling of AAV vector-induced inflammatory and immune response markers in the presence or absence UPR inhibitor during hepatic gene transfer <i>in vivo</i>.

<p>Hepatic gene expression of various inflammatory cytokines in the scAAV2 injected BALB/c mice was measured 24 hours post vector administration. Genes which are significantly different between (2 fold, p<0.05) between mice that received AAV2 and metformin compared to the vector administered group alone, are shown.</p