Journal: eneuro

Article Title: TrkB Signaling Influences Gene Expression in Cortistatin-Expressing Interneurons

doi: 10.1523/eneuro.0310-19.2019

Figure Lengend Snippet: Figure 3. Loss of TrkB signaling in Cort neurons alters the expression of genes important for calcium homeostasis and axon development. A, Breeding strategy used to obtain control CortCre; Rpl22HA and experimental CortCre;TrkBflox/flox;Rpl22HA mice. B, Validation of Ribotag allele expression in cortistatin cells of control and CortCre;TrkBflox/flox;Rpl22HA mice by qPCR of Cort as well as Gad, Gfap, and Bdnf exon IV (n 6 per genotype, Student’s unpaired t test; data are presented as the mean SEM: p 0.05 p 0.01, p 0.001, p 0.0001 vs control). C, Volcano plot of RNA-seq results with CortCre;TrkBflox/flox;Rpl22HA IP versus CortCre;Rpl22HA IP log2 fold change against log10 p value. Orange dots represent genes that are significantly different in CortCre;TrkBflox/flox;Rpl22HA IP versus CortCre; Rpl22HA IP, including Wt1, Calb1, Lgals1, Trpc6, Syt6, and Gng4. Green dots represent nonsignificant genes. See Extended Data Figure 3-1. D, GO terms in the molecular function, biological processes, and cellular component categories for genes enriched and de-enriched in Cort neurons following removal of TrkB and disruption of BDNF–TrkB signaling. See Extended Data Figures 3-2, 3-3, and 3-4.

Article Snippet: TaqMan probes were commercially available from Thermo Fisher Scientific (Gad1 Mm00725661_s1, Cort Mm00432631_m1, Gfap Mm01253033_m1, Wt1 Mm01337048_m1; Cxcr4 Mm01292123_m1; Calb1 Mm00486647_m1; Lgals1 Mm00839408_g1; Trpc6 Mm01176083_m1; Syt6 Mm04932997_m1; Gng4 Mm00772342_m1; Ttc9b Mm01176446_m1; S100a10 Mm00501458_g1; Nxph1 Mm01165166_m1; and Syt2 Mm00436864_m1; Gsn Mm00456679_m1) or as described in the study by Martinowich et al. (2011).

Techniques: Expressing, Control, Biomarker Discovery, RNA Sequencing, Disruption

Journal: eneuro

Article Title: TrkB Signaling Influences Gene Expression in Cortistatin-Expressing Interneurons

doi: 10.1523/eneuro.0310-19.2019

Figure Lengend Snippet: Figure 4. Validation of select targets from Control versus CortCre;TrkBflox/flox RNA-seq using qPCR and single-molecule fluorescence in situ hybridization. A, qPCR analysis validating select genes (Trpc6, Calb1, Lgals1, Wt1, Syt6, Gng4) found to be differentially expressed in CortCre;TrkBflox/flox IP versus control IP RNA-seq data (n 6 per genotype, Student’s unpaired t test; data are presented as the mean SEM: p 0.01, p 0.001, p 0.0001 vs control). B, Quantification of Wt1 transcripts in Cre positive cells of CortCre;TrkBflox/flox and control mice. C, D, Confocal z-projections of Cre and Wt1 transcripts in the cortex from P21 Control (C) and CortCre;TrkBflox/flox (D) mice visualized with RNAscope in situ hybridization. Wt1 transcripts (green) are more enriched in Cort neurons of CortCre;TrkBflox/flox than of Control mice. Inset depicts higher magnification of nuclei highlighted by arrows. Scale bars, C, D, 10 m.

Article Snippet: TaqMan probes were commercially available from Thermo Fisher Scientific (Gad1 Mm00725661_s1, Cort Mm00432631_m1, Gfap Mm01253033_m1, Wt1 Mm01337048_m1; Cxcr4 Mm01292123_m1; Calb1 Mm00486647_m1; Lgals1 Mm00839408_g1; Trpc6 Mm01176083_m1; Syt6 Mm04932997_m1; Gng4 Mm00772342_m1; Ttc9b Mm01176446_m1; S100a10 Mm00501458_g1; Nxph1 Mm01165166_m1; and Syt2 Mm00436864_m1; Gsn Mm00456679_m1) or as described in the study by Martinowich et al. (2011).

Techniques: Biomarker Discovery, Control, RNA Sequencing, Fluorescence, In Situ Hybridization, RNAscope

Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet: (A–F) Immunohistochemistry technique for capturing SNPH intrusion in PC dendrites. Shown is SNPH (green), Syt2 (red), and Calbindin (blue) labeling in 3.5-month-old WT (A, C, and E) and Shi (B, D, and F) mice. Scale bar, 10 μm. (G and H) High magnification of the maximum intensity projection image from the z stack through dendritic regions of WT (G) and Shi (H). (I and J) Orthogonal (slice) view of SNPH punctum (indicated by arrows in panels G and H) in the dendritic region from WT (I) and Shi (J) in x-z and y-z orientations, respectively. (K) Quantification of percentage area occupied by SNPH within the dendritic volume from 3 mice of each group. Data are shown as mean ± SEM. *p < 0.05. (L–N) Capturing SNPH intrusion by pre-tagging dendritic mitochondria in vivo using viral transduction. (L) Technique to selectively transduce PCs with AAV-Mito-mCherry. (M) Demonstration of successful pre-tagging of mitochondria in dendrites of a single PC by Calbindin staining. Scale bar, 10 μm. (N) Demonstration of how pre-tagged dendritic mitochondria in PCs are used to capture SNPH intrusion by 3D rotation. (O and P) Single dendritic tree in WT (O) or Shi (P) pre-tagged with Mito-mCherry (red) and SNPH intrusion (green) captured by co-rotation with Mito-mCherry. Merged images show the fraction of dendritic mitochondria anchored by intruded SNPH (yellow). (Q and R) Percentage of PCs with SNPH intrusions (Q) and percentage of SNPH bound to mitochondria per dendritic tree (R) from WT (n = 73) and Shi (n = 106) PCs. Data are shown as mean ± SEM. ***p < 0.005.

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Immunohistochemistry, Labeling, In Vivo, Transduction, Staining

Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet: (A and B) Representative images of lentivirally transduced GFP-SNPH (1–469) (A) and GFP-SNPH (B) in PCs of SNPH-KO mice injected with saline (no harmaline, vehicle only). (C-H) Effect of harmaline on GFP-SNPH (1–469)-transduced (C) and GFP-SNPH-transduced (F) PC dendrites. Degenerating dendrites in GFP-SNPH-transduced PCs can be seen in (F). Also shown is Calbindin labeling of GFP SNPH (1–469) (D) and GFP-SNPH (G) from (C) and (F). Merged images of GFP SNPH (1–469) and GFP-SNPH with Calbindin are shown in (E) and (H), respectively. (I–K) Representative image of a harmaline-induced degenerating PC (white arrow in I) transduced with GFP-SNPH. Calbindin staining from the same section is shown in (J), whereas a merged image is shown in (K). Scale bars, 20 μm. (L) Quantification of dendritic shrinkage in GFP-SNPH (1–469)- and GFP-SNPH-transduced PCs in the absence (n = 3 mice, vehicle only) or presence of harmaline (n = 5 mice). Data are shown as mean ± SEM. ***p < 0.001.

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Injection, Saline, Labeling, Transduction, Staining

Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet:

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Imaging

Journal: Stem Cells Translational Medicine

Article Title: In vivo survival and differentiation of Friedreich ataxia iPSC ‐derived sensory neurons transplanted in the adult dorsal root ganglia

doi: 10.1002/sctm.20-0334

Figure Lengend Snippet: Expression of markers of nociceptors, proprioceptors, and mechanoreceptors subtypes in FRDA iPSC‐derived sensory neuronal cultures. Differentiated cultures show cluster of neurons expressing NF200 (A). Neurons co‐expressing both PRPH (B, D) and PV (C, D) and PRPH (E, G) and SPP1 (F, G) were found. Expression of FAM19A1 (H) and STT (I) was observed. Group of neurons co‐expressing PLXNC1 (J, red) and CGRP (K, green). Cell positive for CALB1 (L) was detected. Co‐expression of NECAB2 (M, P, green) and TRKB (N, P) was also observed in differentiated cultures. Cells positive for FAM19A1 (Q, T) and TRKA (R, T). C‐LTMRs neuron positive both for PRPH (U, X, red, arrowed) and TH (V, X, green, arrowed). Nuclei are shown in blue (DAPI). Scale bars = (A, T, J, K) 20 μm, (D, G, H, I, L, P) 10 μm, (X) 50 μm

Article Snippet: The specific probes (Life Technologies) that have been used are as follows: BRN3A (POU4F1) (Hs00366711_m1), CALB1 (Hs010077197_m1), ELF1 (Hs00152844_m1), FAM19A1 (Hs00405421_m1), GAPDH (Hs02758991_g1), ( HMBS (Hs00609297_m1), ISLET1 (Hs00158126_m1), LDHB (Hs00929956_m1), NECAB2 (Hs00332810_m1), TRKA (NTRK1) (Hs01021011_m1), TRKB ( NTRK2) (Hs00178811_m1), TRKC (NTRK3) (Hs00176797_m1), PLXNC1 (Hs00194968_m1), PRPH (Hs00986946_g1), PVALB (Hs00161045_m1), RET (Hs01120030_m1), S100β (Hs00902901_m1), SPP1 (Hs00959010_m1), STT (Hs00356144_m1), TAC1 (Hs00243225_m1), TRPV1 (Hs00218912_m1), TH (Hs00165941_m1), VGLUT3 (Hs00900423_m1).

Techniques: Expressing, Derivative Assay