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UNLABELLED Among the known genetic risk factors for Parkinson disease,mutations in GBA1,the gene responsible for the lysosomal disorder Gaucher disease,are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics,we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease,two with and two without parkinsonism,and one patient with Type 2 (acute neuronopathic) Gaucher disease,and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine,demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein,a protein present as aggregates in Parkinson disease and related synucleinopathies,were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607,a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme,restored glucocerebrosidase activity and protein levels,and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons,indicating its potential for treating neuronopathic Gaucher disease. In addition,NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism,suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease,dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity,reduced lysosomal glucocerebrosidase levels,and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype,the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone,which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition,the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons,indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease. View Publication

Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific,as they reduce infection rates in elective surgery,but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably,AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery,consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly,AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells,which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms. View Publication

Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis,a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype-phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal,we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the gold-standard but relatively low-throughput Ussing chamber. Moreover using this approach we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures. View Publication

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans,the number of extracellular vesicles (EVs) increased acutely. In humans,EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins,and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with,and mobilized,splenic monocytes in otherwise naive,healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets,which regulate relevant cellular functions (e.g.,proliferation and cell movement). Furthermore,gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs,EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes,including the negative regulator of cell motility,plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI. View Publication

Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2,ApoE3,and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD),ApoE3 is neutral,and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis,however,remains unclear. Using ES-cell-derived human neurons,we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4< ApoE3< ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK),a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation,stimulating the transcription factor AP-1,which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis. View Publication

BACKGROUND There has been increasing interest recently in the plasticity of mesenchymal stem cells (MSCs) and their potential to differentiate into neural lineages. To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages,we have differentiated MSCs into neural lineages using different combinations of growth factors. Based on previous studies of the roles of insulin-like growth factor 1 (IGF-1) in neural stem cell isolation in the laboratory,we hypothesized that IGF-1 can enhance proliferation and reduce apoptosis in neural progenitor-like cells (NPCs) during differentiation of MSCs into NCPs.We induced MSCs differentiation under four different combinations of growth factors: (A) EGF%+%bFGF,(B) EGF%+%bFGF%+%IGF-1,(C) EGF%+%bFGF%+%LIF,(D) EGF%+%bFGF%+%BDNF,and (E) without growth factors,as a negative control. The neurospheres formed were characterized by immunofluorescence staining against nestin,and the expression was measured by flow cytometry. Cell proliferation and apoptosis were also studied by MTS and Annexin V assay,respectively,at three different time intervals (24 hr,3 days,and 5 days). The neurospheres formed in the four groups were then terminally differentiated into neuron and glial cells. RESULTS The four derived NPCs showed a significantly higher expression of nestin than was shown by the negative control. Among the groups treated with growth factors,NPCs treated with IGF-1 showed the highest expression of nestin. Furthermore,NPCs derived using IGF-1 exhibited the highest cell proliferation and cell survival among the treated groups. The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups. CONCLUSIONS Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the NPCs. This information will be beneficial in the long run for improving both cell-based and cell-free therapy for neurodegenerative diseases. View Publication

Recently there has been growing interest in the differentiation of mesenchymal stem cells (MSCs) into neural lineages. Research suggests that MSCs can be differentiated into neural progenitor-like cells (NPCs) under the specific influence of paracrine factors particularly epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Our recent research has found that the addition of insulin-like growth factor 1 (IGF-1) with the combination of the EGF and bFGF could significantly improve the growth and survivability of MSC-derived NPCs. To unravel the molecular mechanism of the improved differentiation we compared the microRNA expression profiles of the differentiation under various combinations of growth factors. MSCs were differentiated into neural lineage in 3 groups; Group A (EGF + bFGF),Group B (EGF + bFGF + IGF-1),and Group C (without growth factor). Regulated microRNAs during the early differentiation were identified by detailed microRNA profiling using Affymetrix GeneChip version 2.0 at three time intervals (day 1,day 3 and day 5). The data were deposited in the Gene Expression Omnibus,series GSE60060. View Publication

Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO),a chemical modulator of small-/intermediate-conductance Ca(2+)-activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs),we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs,timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs,including an increase in nodal- and atrial-like phenotypes. However,our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and,subsequently,CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO,presumably via an SK-independent mechanism. Together,EBIO did not promote cardiogenic differentiation of PSCs,opposing previous findings,but triggered lineage-selective survival at a cardiac progenitor stage,which we propose as a pharmacological strategy to modulate CM subtype composition. View Publication

BACKGROUND Glioblastoma (GBM),being a highly vascularised and locally invasive tumour,is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks. METHODS Cell viability,endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures. RESULTS An endothelial/GBM cell viability inhibitory effect,as well as an anti-angiogenic and anti-invasive response,to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020,P=0.030),anti-angiogenic (P=0.040,P<0.0001) and pro-apoptotic (P=0.0083,P=0.0149) response was observed when combined treatment was compared with single gossypol/TMZ treatment response,respectively. GBM cell line and patient-specific response to gossypol/TMZ treatment was observed. CONCLUSIONS Our results indicate that response to a combined gossypol/TMZ treatment is related to inhibition of tumour-associated angiogenesis,invasion and proliferation and warrants further investigation as a novel targeted GBM treatment strategy. View Publication

Although many previous investigations have studied how mercury compounds cause cell death,sub-cytotoxic levels may affect mechanisms essential for the proper development of the nervous system. The present study investigates whether low doses of methylmercury (MeHg) and mercury chloride (HgCl2) can modulate the activity of JAK/STAT signaling,a pathway that promotes gliogenesis. We report that sub-cytotoxic doses of MeHg enhance ciliary neurotrophic factor (CNTF) evoked STAT3 phosphorylation in human SH-SY5Y neuroblastoma and mouse cortical neural progenitor cells (NPCs). This effect is specific for MeHg,since HgCl2 fails to enhance JAK/STAT signaling. Exposing NPCs to these low doses of MeHg (30-300nM) enhances CNTF-induced expression of STAT3-target genes such as glial fibrillary acidic protein (GFAP) and suppressors of cytokine signaling 3 (SOCS3),and increases the proportion of cells expressing GFAP following 2 days of differentiation. Higher,near-cytotoxic concentrations of MeHg and HgCl2 inhibit STAT3 phosphorylation and lead to increased production of superoxide. Lower concentrations of MeHg effective in enhancing JAK/STAT signaling (30nM) do not result in a detectable increase in superoxide nor increased expression of the oxidant-responsive genes,heme oxygenase 1,heat shock protein A5 and sirtuin 1. These findings suggest that low concentrations of MeHg inappropriately enhance STAT3 phosphorylation and glial differentiation,and that the mechanism causing this enhancement is distinct from the reactive oxygen species-associated cell death observed at higher concentrations of MeHg and HgCl2. View Publication