Journal: PLoS ONE

Article Title: Genetic Regulation of α-Synuclein mRNA Expression in Various Human Brain Tissues

doi: 10.1371/journal.pone.0007480

Figure Lengend Snippet: Fold levels of SNCA -mRNA were assayed by real-time RT-PCR using TaqMan technology and calculated relative to the geometric mean of SYP- and ENO2- mRNAs reference control using the 2 −ΔCt method. The bar graph presents the average of SNCA -mRNA fold expression (mean±S.E.M) of the 7 subjects for each brain region. SN-substantia nigra; TC-temporal cortex; FC- frontal cortex.

Article Snippet: Of note is that three internal controls were compared: the neuronal specific genes Enolase 2 ( ENO2 Hs00157360_m1) and synaptophysin ( SYP Hs00300531_m1) and the house keeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH Hs00999905_m1).

Techniques: Quantitative RT-PCR, Control, Expressing

Journal: PLoS ONE

Article Title: Genetic Regulation of α-Synuclein mRNA Expression in Various Human Brain Tissues

doi: 10.1371/journal.pone.0007480

Figure Lengend Snippet: Individuals were genotyped for Rep1. Three brain regions were analyzed: temporal cortex (A), midbrain including SN (B) and frontal cortex (C). In each brain region fold levels of human SNCA -mRNA were assayed by real-time RT-PCR using TaqMan technology and calculated relative to human SYP -mRNA reference control using the 2 −ΔCt method. (A) Analysis of the temporal cortex showed that the protective genotype 259/259 correlates with lower SNCA -mRNA levels then the five other genotypes (P = 0.02). The association trend was confirmed in a subset of samples using also the GAPDH and ENO2 reference genes . (B) In the midbrain including SN the 259/259 correlates, with lower SNCA -mRNA levels. (C) No correlations of Rep1 genotypes with SNCA -mRNA levels were detected in the frontal cortex. For each genotype the box plot represents the analysis performed using all brain samples available from the specific brain region, each of which was analyzed twice independently, each time in duplicate. The average values are presented in ‘X’. The box plot shows the median (horizontal line inside the box) and the 25 th and 75 th percentiles (horizontal borders of the box). The range between the 25th and 75th percentiles is the interquartile-range (IQR). The whiskers show the minimal and maximal values inside the main data body.

Article Snippet: Of note is that three internal controls were compared: the neuronal specific genes Enolase 2 ( ENO2 Hs00157360_m1) and synaptophysin ( SYP Hs00300531_m1) and the house keeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH Hs00999905_m1).

Techniques: Quantitative RT-PCR, Control

Journal: PLoS ONE

Article Title: Genetic Regulation of α-Synuclein mRNA Expression in Various Human Brain Tissues

doi: 10.1371/journal.pone.0007480

Figure Lengend Snippet: Individuals were genotyped for SNP rs356219. Three brain regions were analyzed: temporal cortex (A), midbrain including SN (B) and frontal cortex (C). In each brain region fold levels of human SNCA -mRNA were assayed by real-time RT-PCR using TaqMan technology and calculated relative to human SYP -mRNA reference control using the 2 -ΔCt method. (A) Analysis of the temporal cortex showed that the protective genotype AA correlates with higher SNCA -mRNA levels than the GA and GG genotypes (P = 0.013). The association trend was confirmed in a subset of samples using also the GAPDH and ENO2 reference genes . (B) In the midbrain including SN the AA and AG genotypes correlate with higher SNCA-mRNA levels compared with the GG risk genotype (P<0.05). (C) No correlations of SNP rs356219 genotypes with SNCA -mRNA levels were detected in the frontal cortex. For each genotype the box plot represents the analysis performed using all brain samples available from the specific brain region, each of which was analyzed twice independently, each time in duplicate. The average values are presented in ‘X’. The box plot shows the median (horizontal line inside the box) and the 25 th and 75 th percentiles (horizontal borders of the box). The range between the 25th and 75th percentiles is the interquartile-range (IQR). The whiskers show the minimal and maximal values inside the main data body.

Article Snippet: Of note is that three internal controls were compared: the neuronal specific genes Enolase 2 ( ENO2 Hs00157360_m1) and synaptophysin ( SYP Hs00300531_m1) and the house keeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH Hs00999905_m1).

Techniques: Quantitative RT-PCR, Control

Journal: PLoS ONE

Article Title: Genetic Regulation of α-Synuclein mRNA Expression in Various Human Brain Tissues

doi: 10.1371/journal.pone.0007480

Figure Lengend Snippet: Individuals were genotyped for SNP rs365165. Three brain regions were analyzed: temporal cortex (A), midbrain including SN (B) and frontal cortex (C). In each brain region fold levels of human SNCA -mRNA were assayed by real-time RT-PCR using TaqMan technology and calculated relative to human SYP -mRNA reference control using the 2 -ΔΔCt method. (A) Analysis of the temporal cortex showed that the protective genotype AA correlates with higher SNCA -mRNA levels than the GA and GG genotypes (P<0.05). (B) In the midbrain including SN, the AA and AG genotypes correlate with higher SNCA-mRNA levels compared with the GG risk genotype (P<0.05). (C) No correlations of SNP rs365165 genotypes with SNCA-mRNA levels were detected in the frontal cortex. For each genotype, the box plot represents the analysis performed using all brain samples available from the specific brain region, each of which was analyzed twice independently, each time in duplicate. The average values are presented in ‘X’. The box plot shows the median (horizontal line inside the box) and the 25 th and 75 th percentiles (horizontal borders of the box). The range between the 25th and 75th percentiles is the interquartile-range (IQR). The whiskers show the minimal and maximal values inside the main data body.

Article Snippet: Of note is that three internal controls were compared: the neuronal specific genes Enolase 2 ( ENO2 Hs00157360_m1) and synaptophysin ( SYP Hs00300531_m1) and the house keeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH Hs00999905_m1).

Techniques: Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Astrocytic Regulation of Basal Ganglia Dopamine/D2-Dependent Behaviors

doi: 10.1101/2021.05.11.443394

Figure Lengend Snippet: Protein expression displayed by C57BL6J adult mice of a. Dys1A and b. Dys1C isoforms in the PFC, STR, NAcc, and GPe relative to PFC and normalized to their own β-actin. Dys1A expression is higher in GPe compared to PFC and STR (One-way ANOVA: F 3,16 =5.02; p=0.02). *p<0.05 vs PFC and STR. Dys1C expression is uniform across the selected brain areas (One-way ANOVA: F 3,16 =0.045; p=0.98). c-d. D2 receptor expression in Dys1A+/+ (n9), Dys1A+/- (n10) and Dys1A-/- (n10). Synaptophysin used as cytoplasmic control, and D2 expression normalized on transferrin as loading control. c. Dys1A+/- and Dys1A-/- mice show increased total D2 expression compared to Dys1A+/+ in GPe (One-way ANOVA: F 2,16 =4.93; p=0.021), but not in PFC (F 2,25 =0.724; p=0.49), and STR (One-way ANOVA: F 2,22 =1.44; p=0.26). d. Biotinylation protocol, brain slices treated with biotin to label all surface proteins, precipitated by streptavidin. Dys1A-/- mice had increased expression of D2 receptors on cellular surface compared to Dys1A+/+ littermates in STR and GPe (One-way ANOVA: STR F 2,14 =4.20; p=0.04; GPe F 1,6 =6,18; p=0.04), but not in PFC (One-way ANOVA: F 2,24 =0.44; p=0.64). *p<0.05 vs Dys1A+/+. e-g. Dopamine (DA), DOPAC, and HVA content by HPLC, expressed as ng/mg of tissue in e. PFC, f. STR, and g. GPe dissected from Dys1A+/+ (n4), Dys1A+/- (n4), and Dys1A-/- (n4) littermates. No Dys1A-dependent changes were observed in PFC (One-way ANOVA: F 2,5 =0.23; p=0.80). Dys1A-/- show reduced DA and HVA levels relative to Dys1A+/+ in the STR (One-way ANOVA: F 1,10 =5.17; p=0.05). Dys1A+/- and Dys1A-/- show reduced DA levels than Dys1A+/+ in GPe (F 1,10 =10.44; p=0.02). *p<0.05 vs Dys1A+/+. h. Expression of Dys1A and Dys1C isoforms in postmortem caudate from 22 patients with schizophrenia (Schizophrenia) and 18 matched healthy subjects (Control). No differences were present in non-diagnostic variables (i.e., age, sex, post-mortem interval, pH: Supplementary Fig. S4). Expression of Dys1A, normalized by β-actin, is significantly reduced in the caudate of patients with schizophrenia compared to control subjects (t-test: p=0.02). Dys1C expression is not changed between the two groups (t-test: p=0.71). *p<0.05 vs Control. i. Plotting of β-actin normalized data for Dys1A and Dys1C for all case-control pairs. Each bar indicates the log2 transformed ratio of isoform in a schizophrenia case compared to that in its matched control (i.e. the ratio for one case-control pair). Pair-wise analysis of these ratios (Wilcoxon signed-rank test) showed significant difference between schizophrenia cases and their matched controls for Dys1A (W=154.00; p=0.02), but not for Dys1C (W=162.00; p=1.00). Bar graphs show mean ± s.e.m.

Article Snippet: Antibody used were dopamine D2 receptor (sc- 5303, Santa Cruz Biotechnology, Dallas, TX, USA) and (AB5084P, Millipore), Synaptophysin (sc- 365488, Santa Cruz Biotechnology) and Transferrin Receptor (sc-21011, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Diagnostic Assay, Transformation Assay

Journal: eLife

Article Title: Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord

doi: 10.7554/elife.83108

Figure Lengend Snippet: Figure 4. Serial block-face scanning electron microscopy (SBF-SEM) analyses with COX4-dAPEX2 and SYP-HRP labeling reveal structures and recurrent connections of cerebrospinal fluid-contacting neurons (CSF-cNs). (A, B) Representative light microscopic images of 3, 3'-diaminobenzidine (DAB)- stained spinal cord sections of Pkd2l1-Cre mice with injection of adeno-associated virus (AAV)-Syn-DIO-COX4-dAPEX2 (A) or AAV-Syn-DIO-SYP-HRP (B). (C) A low-magnification electron microscopic image of the cervical cord around the central canal (Cc) in Pkd2l1-Cre mice injected with AAV-Syn-DIO-

Article Snippet: AAV plasmids were generated as follows: for pAAV- Syn- mCherry, mCherry of pmCherry- C1 plasmid (Clontech) was subcloned into pAAV- hSyn- EGFP (Addgene, #50465) in a substitution of EGFP; for pAAV- Syn- DIO- COX4- dAPEX2 and pAAV- Syn- DIO- SYP- HRP, COX4- dAPEX2 and SYP- HRP derived from pAAV- EF1a- COX4- dAPEX2 (Addgene, #117176) and pAAV- EF1a- SYP- HRP (Addgene, #117185) were subcloned into pAAV- Syn- DIO- MCS that had multicloning sites (MCS) between the DIO site of pAAV- Syn- DIO- mCherry (Addgene, #50459) in a substitution of mCherry; and for pAAV- Syn- DIOChR2- mCherry, ChR2(H134R)- mCherry derived from pAAV- CAG- ChR2(H134R)- mCherry (Addgene, #100054) was subcloned into pAAV- Syn- DIO- MCS plasmid. pAAV- hSyn- EGFP (Addgene, #50465), pAAV- CAG- EGFP (pENN.AAV.CB7.CI.eGFP.WPRE.rBG; Penn vector core, #AV- 1- PV1963), and pAAVSyn- DIO- mCherry (Addgene, #50459) were further used for AAV production.

Techniques: Blocking Assay, Electron Microscopy, Labeling, Staining, Injection, Virus

Journal: Molecular Neurodegeneration

Article Title: Regulatory T cells limit age-associated retinal inflammation and neurodegeneration

doi: 10.1186/s13024-024-00724-w

Figure Lengend Snippet: Antibodies table

Article Snippet: Guinea Pig Anti-Synaptophysin , AGP-144 , Alomone , Guinea Pig , 1:150.

Techniques: Binding Assay, Recombinant, Functional Assay