Inas Helwa

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40–150 nm). The three kits, though, produced a significantly higher yield (80–300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.

Neutral Sphingomyelinase-2 Deficiency Ameliorates Alzheimer's Disease Pathology and Improves Cognition in the 5XFAD Mouse

Recent evidence implicates exosomes in the aggregation of Aβ and spreading of tau in Alzheimers disease. In neural cells, exosome formation can be blocked by inhibition or silencing of neutral sphingomyelinase-2 (nSMase2). We generated genetically nSMase2-deficient 5XFAD mice (fro;5XFAD) to assess AD-related pathology in a mouse model with consistently reduced ceramide generation. We conducted in vitro assays to assess Aβ42 aggregation and glial clearance with and without exosomes isolated by ultracentrifugation and determined exosome-induced amyloid aggregation by particle counting. We analyzed brain exosome content, amyloid plaque formation, neuronal degeneration, sphingolipid, Aβ42 and phospho-tau levels, and memory-related behaviors in 5XFAD versus fro;5XFAD mice using contextual and cued fear conditioning. Astrocyte-derived exosomes accelerated aggregation of Aβ42 and blocked glial clearance of Aβ42 in vitro. Aβ42 aggregates were colocalized with extracellular ceramide in vitro using a bifunctional ceramide analog preloaded into exosomes and in vivo using anticeramide IgG, implicating ceramide-enriched exosomes in plaque formation. Compared with 5XFAD mice, the fro;5XFAD mice had reduced brain exosomes, ceramide levels, serum anticeramide IgG, glial activation, total Aβ42 and plaque burden, tau phosphorylation, and improved cognition in a fear-conditioned learning task. Ceramide-enriched exosomes appear to exacerbate AD-related brain pathology by promoting the aggregation of Aβ. Reduction of exosome secretion by nSMase2 loss of function improves pathology and cognition in the 5XFAD mouse model. SIGNIFICANCE STATEMENT We present for the first time evidence, using Alzheimers disease (AD) model mice deficient in neural exosome secretion due to lack of neutral sphingomyelinase-2 function, that ceramide-enriched exosomes exacerbate AD-related pathologies and cognitive deficits. Our results provide rationale to pursue a means of inhibiting exosome secretion as a potential therapy for individuals at risk for developing AD.

Screening of the Seed Region of MIR184 in Keratoconus Patients from Saudi Arabia

Micro-RNAs (miRNAs) are regulators of gene expression that control various biological processes. The role of many identified miRNAs is not yet resolved. Recent evidence suggests that miRNA mutations and/or misexpression may contribute to genetic disorders. Point mutations in the seed region of MIR184 have been recently identified in Keratoconus (KC) patients with or without other corneal and lens abnormalities. We investigated mutations within MIR184 in KC patients from Saudi Arabia and examined the relative expression of miR-184 and miR-205 in human cornea. Ethnically matched KC cases (n = 134) were recruited and sequencing was performed using PCR-based Sanger sequencing and analyzed using the Sequencher 5.2 software. Expression of miR-184 and miR-205 was profiled in postmortem unaffected ocular tissues obtained from donors with no history of ocular diseases. miR-184 expression was 15-fold higher than that of miR-205 in cornea samples. No mutation(s) within the screened genomic region of MIR184 in KC cases was detected. This suggests that mutation in MIR184 is a rare cause of KC alone and may be more relevant to cases of KC associated with other ocular abnormalities. The increased expression of miR-184 versus miR-205 in normal cornea samples implies a possible role of miR184 in cornea development and/or corneal diseases.

The Antipsoriatic Agent Monomethylfumarate Has Antiproliferative, Prodifferentiative, and Anti-Inflammatory Effects on Keratinocytes

Monomethylfumarate (MMF) is thought to be the bioactive ingredient of the drug Fumaderm (Biogen Idec, Cambridge, MA), licensed in Germany since 1994 for the treatment of moderate-to-severe psoriasis. Psoriasis is a common inflammatory hyperproliferative skin disorder that involves cross-talk between different cell types, including immune cells and keratinocytes. Psoriatic lesions are characterized by hyperproliferation, aberrant differentiation, and inflammation, with the psoriatic cytokine network maintained by communication between immune cells and keratinocytes. Recently, there is increasing evidence regarding the pivotal role of keratinocytes in mediating the disease process, and these cells can be regarded as safe therapeutic targets. From the data available on human subjects treated with Fumaderm, MMF is an effective antipsoriatic agent with known effects on immune cells. However, little is known about its direct effects on keratinocytes. We hypothesized that MMF has direct antiproliferative, prodifferentiative, and anti-inflammatory effects on keratinocytes. Indeed, MMF dose-dependently inhibited [3H]thymidine incorporation into DNA, indicating a direct antiproliferative action on keratinocytes. MMF significantly increased the protein level of keratin 10, the early keratinocyte differentiation marker, and the activity of transglutaminase, a late differentiation marker. These results are consistent with an ability of MMF to promote keratinocyte differentiation and inhibit proliferation, thereby improving psoriatic lesions. In 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced keratinocytes, MMF significantly inhibited the expression of the proinflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-6, and interleukin-1α as well as the production of TNFα. Our results support the notion that MMF has direct antiproliferative, prodifferentiative, and anti-inflammatory effects on keratinocytes, highlighting its potential use as a multifactorial antipsoriatic agent.

Molecular and Histopathological Changes Associated with Keratoconus

Keratoconus (KC) is a corneal thinning disorder that leads to loss of visual acuity through ectasia, opacity, and irregular astigmatism. It is one of the leading indicators for corneal transplantation in the Western countries. KC usually starts at puberty and progresses until the third or fourth decade; however its progression differs among patients. In the keratoconic cornea, all layers except the endothelium have been shown to have histopathological structural changes. Despite numerous studies in the last several decades, the mechanisms of KC development and progression remain unclear. Both genetic and environmental factors may contribute to the pathogenesis of KC. Many previous articles have reviewed the genetic aspects of KC, but in this review we summarize the histopathological features of different layers of cornea and discuss the differentially expressed proteins in the KC-affected cornea. This summary will help emphasize the major molecular defects in KC and identify additional research areas related to KC, potentially opening up possibilities for novel methods of KC prevention and therapeutic intervention.

miRNA Profile in Three Different Normal Human Ocular Tissues by miRNA-Seq

Purpose Because microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human ciliary body (CB), cornea, and trabecular meshwork (TM) using miRNA-Seq to provide a foundation for better understanding of miRNA function and disease involvement in these tissues. Methods Total RNAs were extracted from seven normal human CB, seven cornea, and seven TM samples using mirVana total RNA isolation kit. miRNA-Seq was done with Illumina MiSeq. Bowtie software was used to trim and align generated sequence reads, and only exact matches to mature miRNAs from miRBase were included. The miRTarBase database was used to analyze miRNA target interactions, and the expression of five selected miRNAs was validated using droplet digital PCR (ddPCR). Results Using the miRNA extracted from 21 human samples, we found 378 miRNAs collectively expressed, of which the 11 most abundant miRNAs represented 80% of the total normalized reads. We also identified uniquely expressed miRNAs, of which five share 18 highly validated gene targets, and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we validated the expression profile of five miRNAs from miRNA-Seq. Conclusions For the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Defining the relative expression of miRNAs in nondiseased eye tissues could help uncover changes in miRNA expression that accompany diseases such as glaucoma and keratoconus.

A Common Variant in MIR182 Is Associated With Primary Open-Angle Glaucoma in the NEIGHBORHOOD ConsortiumAssociation of miR-182 and POAG in NEIGHBORHOOD

Purpose Noncoding microRNAs (miRNAs) have been implicated in the pathogenesis of glaucoma. We aimed to identify common variants in miRNA coding genes (MIR) associated with primary open-angle glaucoma (POAG). Methods Using the NEIGHBORHOOD data set (3853 cases/33,480 controls with European ancestry), we first assessed the relation between 85 variants in 76 MIR genes and overall POAG. Subtype-specific analyses were performed in high-tension glaucoma (HTG) and normal-tension glaucoma subsets. Second, we examined the expression of miR-182, which was associated with POAG, in postmortem human ocular tissues (ciliary body, cornea, retina, and trabecular meshwork [TM]), using miRNA sequencing (miRNA-Seq) and droplet digital PCR (ddPCR). Third, miR-182 expression was also examined in human aqueous humor (AH) by using miRNA-Seq. Fourth, exosomes secreted from primary human TM cells were examined for miR-182 expression by using miRNA-Seq. Fifth, using ddPCR we compared miR-182 expression in AH between five HTG cases and five controls. Results Only rs76481776 in MIR182 gene was associated with POAG after adjustment for multiple comparisons (odds ratio [OR] = 1.23, 95% confidence interval [CI]: 1.11–1.42, P = 0.0002). Subtype analysis indicated that the association was primarily in the HTG subset (OR = 1.26, 95% CI: 1.08–1.47, P = 0.004). The risk allele T has been associated with elevated miR-182 expression in vitro. Data from ddPCR and miRNA-Seq confirmed miR-182 expression in all examined ocular tissues and TM-derived exosomes. Interestingly, miR-182 expression in AH was 2-fold higher in HTG patients than nonglaucoma controls (P = 0.03) without controlling for medication treatment. Conclusions Our integrative study is the first to associate rs76481776 with POAG via elevated miR-182 expression.

Anti-Psoriatic Drug Monomethylfumarate Increases Nuclear Factor Erythroid 2-Related Factor 2 Levels and Induces Aquaporin-3 mRNA and Protein Expression

Oxidative stress contributes to inflammatory skin diseases, including psoriasis. Monomethylfumarate (MMF) is an antipsoriatic agent with a poorly understood mechanism of action. In other cell types MMF increases the expression of nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor that regulates cellular antioxidant responses, to reduce oxidative stress like that observed in inflammatory disorders such as multiple sclerosis. We tested the hypothesis that MMF enhances Nrf2 activity in keratinocytes, thereby improving their capacity to counteract environmental stresses. We used Western analysis, immunofluorescence, and real-time quantitative reverse-transcription polymerase chain reaction to examine the effect of MMF on the expression of Nrf2 and its targets. We also measured intracellular reactive oxygen species (ROS) levels following MMF treatment. Our data show that MMF increased total and nuclear Nrf2 levels in primary mouse keratinocytes and enhanced mRNA expression of several Nrf2-downstream effectors, including heme oxygenase-1 and peroxiredoxin-6. Moreover, MMF treatment attenuated the generation of ROS following hydrogen peroxide treatment. On the other hand, the expression and membranous localization of aquaporin-3 (AQP3), a glycerol channel implicated in keratinocyte differentiation, was stimulated by MMF, which also enhanced keratinocyte glycerol uptake. The Nrf2 activator sulforaphane also increased AQP3 levels, suggesting that AQP3 expression may be regulated by Nrf2. We show for the first time that MMF stimulates Nrf2 and AQP3 expression and function/activity in keratinocytes. This effect may account, in part, for the previously observed ability of MMF to inhibit proliferation and inflammatory mediator production and promote differentiation in keratinocytes and to treat psoriasis.

Regulation of the Glycerol Transporter, Aquaporin-3, by Histone Deacetylase-3 and p53 in Keratinocytes

Aquaporin- (AQP) 3, a water and glycerol channel, plays an important role in epidermal function, with studies showing its involvement in keratinocyte proliferation, differentiation, and migration and in epidermal wound healing and barrier repair. Increasing speculation about the use of histone deacetylase (HDAC) inhibitors to treat skin diseases led us to investigate HDACs role in the regulation of AQP3. The broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid induced AQP3 mRNA and protein expression in a dose- and time-dependent manner in normal keratinocytes. The SAHA-induced increase in AQP3 levels resulted in enhanced [3H]glycerol uptake in normal but not in AQP3-knockout keratinocytes, confirming that the expressed AQP3 was functional. Use of HDAC inhibitors with different specificities limited our exploration of the responsible HDAC member to HDAC1, HDAC2, or HDAC3. Cre-recombinase-mediated knockdown and overexpression of HDAC3 suggested a role for HDAC3 in suppressing AQP3 expression basally. Further investigation implicated p53 as a transcription factor involved in regulating HDAC inhibitor-induced AQP3 expression. Thus, our study supports the regulation of AQP3 expression by HDAC3 and p53. Because suberoylanilide hydroxamic acid is already approved to treat cutaneous T-cell lymphoma, it could potentially be used as a therapy for skin diseases like psoriasis, where AQP3 is abnormally expressed.

Protein kinase D1 deficiency promotes differentiation in epidermal keratinocytes

BACKGROUND Protein kinase D (PKD or PKD1) is a serine/threonine protein kinase that has been shown to play a role in a variety of cellular processes; however, the function of PKD1 in the skin has not been fully investigated. The balance between proliferation and differentiation processes in the predominant cells of the epidermis, the keratinocytes, is essential for normal skin function. OBJECTIVE To investigate the effect of PKD1 deficiency on proliferation and differentiation of epidermal keratinocytes. METHODS We utilized a floxed PKD1 mouse model such that infecting epidermal keratinocytes derived from these mice with an adenovirus expressing Cre-recombinase allowed us to determine the effect of PKD1 gene loss in vitro. Proliferation and differentiation were monitored using qRT-PCR, Western blot, transglutaminase activity assays, [3H]thymidine incorporation into DNA and cell cycle analysis. RESULTS A significant decrease in PKD1 mRNA and protein levels was achieved in adenoviral Cre-recombinase-infected cells. Deficiency of PKD1 resulted in significant increases in the mRNA and protein expression of various differentiation markers such as loricrin, involucrin, and keratin 10 either basally and/or upon stimulation of differentiation. PKD1-deficient keratinocytes also showed an increase in transglutaminase expression and activity, indicating an anti-differentiative role of PKD1. Furthermore, the PKD1-deficient keratinocytes exhibited decreased proliferation. However, PKD1 loss had no effect on stem cell marker expression. CONCLUSIONS Cre-recombinase-mediated knockdown represents an additional approach demonstrating that PKD1 is an anti-differentiative, pro-proliferative signal in mouse keratinocytes.