Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: RNA-seq identifies altered neurodevelopmental genes and functions. (a) IPA analysis of DEGs showing the top five functional categories having differential expression of genes related to development (based on −log(P) calculated by Fisher’s exact test. (b) Gene network showing interactions of DEGs, colored in yellow (upregulation) and blue (downregulation), involved in neuronal development and relevant to HD pathogenesis. Several key regulatory pathways have been added to show interactions and possible contribution to the findings, including REST and miR-124, which are predicted upstream regulators with z-scores of 4.038 and −3.638, respectively. HTT is highlighted in orange and connects several critical gene pathways. NEUROD1 manifests as a major node. See Supplementary Figure 2c for symbol legend.

Article Snippet: Antibodies for detection of NEUROD1, CALB1, and CAMKIV were purchased from Cell Signaling: NeuroD (D90G12) rabbit mAb: cat# 7019, 1:1,000 dilution (for WB validation, see https://www.cellsignal.com/products/primary-antibodies/neurod-d90g12-rabbit-mab/7019 ); Calbindin (D1I4Q) XP rabbit mAb: cat# 13176, 1:1,500 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/calbindin-d1i4q-xp-rabbit-mab/13176?_=1487290902164&Ntt=13176&tahead=true ) 80 – 82 ; CaMK IV: rabbit, cat# 4032, 1:1,000 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/camkiv-antibody/4032 ; Product Citations) 83 , 84 .

Techniques: RNA Sequencing, Functional Assay, Quantitative Proteomics

Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: Gene expression changes reflect altered striatal development. (a) Heat map of genes common to mouse microarray analysis and human RNA-seq data. Values are in s.d. normalized by gene separately by set (human and mouse) (yellow, +3; blue, −3). Hierarchical clustering analyses with average linkage for sample, and genes are displayed with dendrograms; clusters were obtained automatically with Genesis Software. Cluster A (violet) includes germinal zone samples and HD human samples, whereas cluster B (green) contains human non-disease samples and mantle zone mouse samples. (b) Gene Ontology (GO) biological process (BP) terms with enrichment of all 679 common genes depicted in the heat map. For GO enrichment, a hypergeometric test was used with P < 0.05 adjusted by Benjamini-Hochberg correction. (c) Summary of the most enriched ‘analyzed network’ result from Metacore using genes from cluster I, which contains NeuroD1. Metacore’s Direct Interactions network was obtained from the 679 common human and mouse genes. The network depicted is filtered to include only transcription factors, protein kinases, receptors with kinase action and ligands. See Supplementary Figure 2c for symbol legend.

Article Snippet: Antibodies for detection of NEUROD1, CALB1, and CAMKIV were purchased from Cell Signaling: NeuroD (D90G12) rabbit mAb: cat# 7019, 1:1,000 dilution (for WB validation, see https://www.cellsignal.com/products/primary-antibodies/neurod-d90g12-rabbit-mab/7019 ); Calbindin (D1I4Q) XP rabbit mAb: cat# 13176, 1:1,500 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/calbindin-d1i4q-xp-rabbit-mab/13176?_=1487290902164&Ntt=13176&tahead=true ) 80 – 82 ; CaMK IV: rabbit, cat# 4032, 1:1,000 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/camkiv-antibody/4032 ; Product Citations) 83 , 84 .

Techniques: Gene Expression, Microarray, RNA Sequencing, Software

Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: Isx-9 induces expression of NEUROD1. (a) Total HTT silencing increases NEUROD1 gene expression in HD iPSC-derived neural cultures. High CAG repeat (109Q) cultures were treated with HTT ASOs and show enhanced NEUROD1 transcript levels. For qPCR analysis, a statistical difference in gene expression was determined using an unpaired two-tailed t-test in GraphPad Prism. HTT: P = 0.0004, t = 8.322, d.f. = 5; NEUROD1: P = 0.0331, t = 5.362, d.f. = 3. (b) NEUROD1 overexpression enhances neuronal gene expression in high CAG repeat lines. HD iPSC-neural cultures were transduced with either human NEUROD1 overexpression lentivirus or pFUGW-eGFP vector control. NEUROD1 overexpression lentivirus (Applied Biological Materials) was used at 1 × 106 infectious units/ml for transduction. Subsequent qPCR demonstrated increased expression of NEUROD1, CALB1 and CAMK4. NEUROD1 (21Q: P = 0.0016, t = 11.03, d.f. = 3; 109Q: P = 0.0002, t = 21.46, d.f. = 3); CALB1 (21Q: P = 0.07; t = 2.456, d.f. = 4; 109Q: P = 0.0065, t = 5.212, d.f. = 4) POU4F2 (21Q: P = 0.775, t = 0.3122, d.f. = 3; 109Q: P = 0.269, t = 1.515, d.f. = 2); CAMK4 (21Q: P = 0.0001, t = 15.33, d.f. = 4; 109Q: P = 0.0004, t = 10.80, d.f. = 4). (c) Isx-9 enhances NEUROD1 expression. qPCR was performed on iPSC-neural cultures after treatment with 20 μM Isx-9. Increased NEUROD1 mRNA was detected in one non-disease (21Q) and both HD (60Q and 109Q) lines. Between vehicle and Isx-9 treatment: 21Q: P = 0.0001, t = 14.89, d.f. = 4; 109Q: P = 0.0006, t = 9.804, d.f. = 4. (d) Isx-9 western analysis. NEUROD1 protein levels increased after treatment with 20 μM Isx-9 in the absence of BDNF. Conversely, BDNF did not increase NEUROD1 protein expression in the absence of Isx-9. Densitometric quantification was normalized to actin. Significance between Isx-9 and untreated cells was determined by one-way ANOVA. 21Q plus Isx-9 (P = 0.000171, n = 4 independent differentiations, d.f. = 1, F = 68.07). 109Q plus Isx-9 (P = 0.0001, n = 4 independent differentiations, d.f. = 4, F = 217.4). Full blots in Supplementary Figure 8. Plots show mean ± s.d.

Article Snippet: Antibodies for detection of NEUROD1, CALB1, and CAMKIV were purchased from Cell Signaling: NeuroD (D90G12) rabbit mAb: cat# 7019, 1:1,000 dilution (for WB validation, see https://www.cellsignal.com/products/primary-antibodies/neurod-d90g12-rabbit-mab/7019 ); Calbindin (D1I4Q) XP rabbit mAb: cat# 13176, 1:1,500 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/calbindin-d1i4q-xp-rabbit-mab/13176?_=1487290902164&Ntt=13176&tahead=true ) 80 – 82 ; CaMK IV: rabbit, cat# 4032, 1:1,000 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/camkiv-antibody/4032 ; Product Citations) 83 , 84 .

Techniques: Expressing, Gene Expression, Derivative Assay, Two Tailed Test, Over Expression, Transduction, Plasmid Preparation, Control, Western Blot

Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: Isx-9 treatment improves CAG repeat-associated phenotypes. (a) Nuclear condensation assay. Differentiated neural cultures were treated with Isx-9 or BDNF. Cell death, by nuclear condensation, was reduced in the HD 109Q line treated with Isx-9. One-way ANOVA (+BDNF +ISX9 versus −BDNF −ISX9, *P = 9 × 10−6; −BDNF −ISX9 versus −BDNF +ISX9, *P = 0.001; n = 7, d.f. = 7, F = 7.011). (b) NEUROD1 knockdown. The HD iPSC-derived neural cells were transduced with lentiviral particles carrying vectors encoding either scrambled (Scrbl) or NEUROD1 shRNA (KD) (shRNA and pFUGW-eGFP lentiviruses at 100 ng/ml for transduction) for 24 h, and then transferred to neural induction medium without additives or supplied with 20 μM Isx-9 for 48 h. NT, non-transduced control. **P < 0.005 versus scrambled control shRNA. One-way ANOVA (P = 1 × 10−10, n = 9, d.f. = 15, F = 11.15). (c) Isx-9 increases survival of HD i-neurons (neural cells derived from iPSCs). Images were used to follow individual cells over time and cell death was recorded to assess the effect of Isx-9 on HD and control cells. Left: cumulative risk of death of the pooled 46Qn1 and 46Qn10 (46Qn1/n10) lines is greater than that of the 18Qn2/n6 controls (hazard ratio (HR) = 1.34, *P = 0.0281). Isx-9 decreased the cumulative risk of death of all 46Qn1/n10 (HR = 0.74, ***P = 7.9 × 10−7) and also of the controls (HR = 0.75, P = 0.08). Control 18Qn2/n6 + DMSO, n = 124 cells, control 18Qn2/n6 + Isx-9, n = 143 cells (4 experiments); 46Qn1/n10 + DMSO, n = 702 cells, 46Qn1/n10 + Isx-9, n = 783 cells (6 experiments). Right: cumulative risk of death of 46Qn1/n10 is significantly greater than that of the control neural cells 18Qn2/6 (HR = 1.3, **P = 0.00209). Isx-9 (20 μM) rescued the survival deficit of 53Qn3 (HR = 0.78, ***P = 9.2 × 10−7). Isx-9 did not significantly change cumulative risk of death for the control Q18n2/6 (HR = 0.97, P = 0.685). All P values are reported from the log rank test, but HR values are from the Cox proportional hazards model. Control Q18n2/6 + DMSO, n = 490 cells, Data shown are all experiments combined into one curve. Individual survival curves are shown in Supplementary Figure 10. (d) HD cells have longer neurite-like process length than controls, and process length is reverted by Isx-9. HD and control iPSCs were differentiated as above. HD cells have longer neurite-like process length than controls; length is restored by Isx-9. HD and control iPSCs were differentiated as above. The HD lines Q46n1, Q46n10, Q53n3, Q53n5, Q109n4 (****P < 0.0001 in all comparisons for these five lines) and Q109n5 (P = 0.0069, P = 0.0018, P < 0.0001) had significantly longer processes compared to control cell lines Q18n2, Q18n6 and Q28n6, respectively. Isx-9 rescued the abnormal increased process length in HD lines Q46n1 (****P < 0.0001), Q46n10 (**P = 0.0067), Q53n3 (****P < 0.0001), Q53n5 (***P = 0.0004), Q109n4 (***P = 0.0005) and Q109n5 (****P < 0.0001) but did not alter process length in control cell lines Q18n2 (P = 0.999), Q18n6 (P = 0.939) and Q28n6 (P > 0.999). Error bars represent the s.d. of the mean.

Article Snippet: Antibodies for detection of NEUROD1, CALB1, and CAMKIV were purchased from Cell Signaling: NeuroD (D90G12) rabbit mAb: cat# 7019, 1:1,000 dilution (for WB validation, see https://www.cellsignal.com/products/primary-antibodies/neurod-d90g12-rabbit-mab/7019 ); Calbindin (D1I4Q) XP rabbit mAb: cat# 13176, 1:1,500 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/calbindin-d1i4q-xp-rabbit-mab/13176?_=1487290902164&Ntt=13176&tahead=true ) 80 – 82 ; CaMK IV: rabbit, cat# 4032, 1:1,000 dilution (for WB validation and references, see https://www.cellsignal.com/products/primary-antibodies/camkiv-antibody/4032 ; Product Citations) 83 , 84 .

Techniques: Knockdown, Derivative Assay, Transduction, shRNA, Control

Journal: Scientific reports

Article Title: Exosomes Derived from HIV-1 Infected DCs Mediate Viral trans-Infection via Fibronectin and Galectin-3.

doi: 10.1038/s41598-017-14817-8

Figure Lengend Snippet: Figure 3. Molecular analysis of exosomes from uninfected and HIV-1 infected DCs (DCex-UN and DCex- HIV). (A) Representative Western blot images showing expression of indicated proteins in the lysates (10 µg) of exosomes derived from uninfected (DCex-UN) or HIV-1 infected (DCex-HIV) DCs. GAPDH served as a loading control. (B) Quantitative analysis of Western blot images of (A); the band intensity of each lane was determined by Image J software and pixel density was calculated. Fold change was determined by considering band intensity of GAPDH in untreated cells as 1 (**p ≤ 0.01, ***p ≤ 0.001, p-values for Annexin A6 – 0.00645, LFA-1 – 0.00047, Integrin αM – 0.00269, Integrin αv – 0.00112, Fibronectin – 0.00051, Tubulin – 0.00028, MHC-II – 0.00408. (C) Representative Western blot images showing expression of HIV-1 proteins in the lysates of exosomes derived from uninfected or HIV-1 infected DCs. GAPDH was used as a loading control. Full- length blots are presented in Supplementary Figure S3. (D) Total RNA was isolated from exosomes derived from uninfected (DCex-UN) or HIV-1 infected (DCex-HIV) DCs and analyzed for the expression HIV-1 genes by qRT-PCR. The data represents mean of triplicates ± SE from three independent experiments.

Article Snippet: CD63, CD9, CD81, HSP70, Integrin αM, Integrin α3, Integrin α5, Annexin A6, TSG101, Alix, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Infection, Western Blot, Expressing, Derivative Assay, Control, Software, Isolation, Quantitative RT-PCR