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: Sections were then incubated with primary antibodies against SNPH (1:250; Abcam), Synaptotagmin2 (1:250; Developmental Studies Hybridoma Bank) and Calbindin (1:250; BosterBio) overnight in PBS containing 0.3% Triton X-100 and 5% normal goat serum.

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: Sections were then incubated with primary antibodies against SNPH (1:250; Abcam), Synaptotagmin2 (1:250; Developmental Studies Hybridoma Bank) and Calbindin (1:250; BosterBio) overnight in PBS containing 0.3% Triton X-100 and 5% normal goat serum.

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: Sections were then incubated with primary antibodies against SNPH (1:250; Abcam), Synaptotagmin2 (1:250; Developmental Studies Hybridoma Bank) and Calbindin (1:250; BosterBio) overnight in PBS containing 0.3% Triton X-100 and 5% normal goat serum.

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

Journal: Neuron

Article Title: Radial glial lineage progression and differential intermediate progenitor amplification underlie striatal compartments and circuit organization

doi: 10.1016/j.neuron.2018.06.021

Figure Lengend Snippet: (A) Schematic of LGE progenitor types, including proliferating neuroepithelial cells (NE), self-renewing and neurogenic radial glial cells (RGS), apical intermediate progenitors (aIPs), and basal intermediate progenitors (bIPs). aIPs include short neural precursors (SNP) and sub-apical progenitors (SAP). These progenitor types are marked by Gsx1/2, Tis21, Ascl1 expression. Tis21 marks neurogenic RGs, aIPs and bIPs. VZ: ventricular zone. SVZ: subventricular zone. MZ: mantle zone.

Article Snippet: Sections were blocked in 10% normal goat serum and 0.1% triton-X100 in 1XPBS, then incubated in primary antibodies (diluted in blocking solution) at 4°C overnight: anti-RFP (rabbit polyclonal antibody, Rockland Pharmaceuticals), anti-MOR1 (rabbit polyclonal, immunostar), anti-Calbindin 28K (rabbit polyclonal, Millipore), anti-CDG1 (rabbit polyclonal, Crittenden et al., 2009 ), anti-Tyrosine Hydroxylase (rabbit polyclonal, Millipore), anti-Ki67 (rabbit polyclonal, Vector), anti-Ascl1/Mash1 (mouse monoclonal, BD biosciences), anti-BrdU (rat monoclonal Accurate Chemical), anti-TuJ1 (mouse monoclonal, Covance), anti-Parvalbumin (mouse monoclonal, Sigma), anti-Somatostatin (rabbit polyclonal, Millipore), anti-ChAT (rabbit polyclonal, Santa Cruz biotechnology), anti-CTIP2 (mouse monoclonal, Abcam), anti-Nkx2.1 (rabbit polyclonal, Santa Cruz biotechnology), anti-Sox2 (human-derived mouse/rat/human monoclonal, R&D Systems).

Techniques: Expressing

Journal: Neuron

Article Title: Radial glial lineage progression and differential intermediate progenitor amplification underlie striatal compartments and circuit organization

doi: 10.1016/j.neuron.2018.06.021

Figure Lengend Snippet: (A) Representative striatum panels of TM time points in Ascl1-CreER;Ai14 mice, including MOR1 co-labeling at peak striosome (E10.5) versus peak matrix (E14.5) cell birth times. Scale bars: 300 µm.

Article Snippet: Sections were blocked in 10% normal goat serum and 0.1% triton-X100 in 1XPBS, then incubated in primary antibodies (diluted in blocking solution) at 4°C overnight: anti-RFP (rabbit polyclonal antibody, Rockland Pharmaceuticals), anti-MOR1 (rabbit polyclonal, immunostar), anti-Calbindin 28K (rabbit polyclonal, Millipore), anti-CDG1 (rabbit polyclonal, Crittenden et al., 2009 ), anti-Tyrosine Hydroxylase (rabbit polyclonal, Millipore), anti-Ki67 (rabbit polyclonal, Vector), anti-Ascl1/Mash1 (mouse monoclonal, BD biosciences), anti-BrdU (rat monoclonal Accurate Chemical), anti-TuJ1 (mouse monoclonal, Covance), anti-Parvalbumin (mouse monoclonal, Sigma), anti-Somatostatin (rabbit polyclonal, Millipore), anti-ChAT (rabbit polyclonal, Santa Cruz biotechnology), anti-CTIP2 (mouse monoclonal, Abcam), anti-Nkx2.1 (rabbit polyclonal, Santa Cruz biotechnology), anti-Sox2 (human-derived mouse/rat/human monoclonal, R&D Systems).

Techniques: Labeling

Journal: Neuron

Article Title: Radial glial lineage progression and differential intermediate progenitor amplification underlie striatal compartments and circuit organization

doi: 10.1016/j.neuron.2018.06.021

Figure Lengend Snippet: Average Number of Progenitors Per Brain in Tis21-CreER;Ai14 Mice Scored as RG, aIP, bIP, and the total number RFP + cells within the ventricular and subventricular zone of the LGE at 8, 48, or 96 hr following E10 TM induction.

Article Snippet: Sections were blocked in 10% normal goat serum and 0.1% triton-X100 in 1XPBS, then incubated in primary antibodies (diluted in blocking solution) at 4°C overnight: anti-RFP (rabbit polyclonal antibody, Rockland Pharmaceuticals), anti-MOR1 (rabbit polyclonal, immunostar), anti-Calbindin 28K (rabbit polyclonal, Millipore), anti-CDG1 (rabbit polyclonal, Crittenden et al., 2009 ), anti-Tyrosine Hydroxylase (rabbit polyclonal, Millipore), anti-Ki67 (rabbit polyclonal, Vector), anti-Ascl1/Mash1 (mouse monoclonal, BD biosciences), anti-BrdU (rat monoclonal Accurate Chemical), anti-TuJ1 (mouse monoclonal, Covance), anti-Parvalbumin (mouse monoclonal, Sigma), anti-Somatostatin (rabbit polyclonal, Millipore), anti-ChAT (rabbit polyclonal, Santa Cruz biotechnology), anti-CTIP2 (mouse monoclonal, Abcam), anti-Nkx2.1 (rabbit polyclonal, Santa Cruz biotechnology), anti-Sox2 (human-derived mouse/rat/human monoclonal, R&D Systems).

Techniques: Pulse Chase

Journal: Neuron

Article Title: Radial glial lineage progression and differential intermediate progenitor amplification underlie striatal compartments and circuit organization

doi: 10.1016/j.neuron.2018.06.021

Figure Lengend Snippet: (A) Schematic of E10.5 short- and long-pulse fate mapping by TM induction in Ascl1-CreER;Ai14 or Dlx1-CreER;Ai14 mice. Ascl1 (red) and Dlx1 expression (darker red) in a LGE coronal section. Striosomes in the striatum (red patches) are depicted in a schematic P28 coronal brain section.

Article Snippet: Sections were blocked in 10% normal goat serum and 0.1% triton-X100 in 1XPBS, then incubated in primary antibodies (diluted in blocking solution) at 4°C overnight: anti-RFP (rabbit polyclonal antibody, Rockland Pharmaceuticals), anti-MOR1 (rabbit polyclonal, immunostar), anti-Calbindin 28K (rabbit polyclonal, Millipore), anti-CDG1 (rabbit polyclonal, Crittenden et al., 2009 ), anti-Tyrosine Hydroxylase (rabbit polyclonal, Millipore), anti-Ki67 (rabbit polyclonal, Vector), anti-Ascl1/Mash1 (mouse monoclonal, BD biosciences), anti-BrdU (rat monoclonal Accurate Chemical), anti-TuJ1 (mouse monoclonal, Covance), anti-Parvalbumin (mouse monoclonal, Sigma), anti-Somatostatin (rabbit polyclonal, Millipore), anti-ChAT (rabbit polyclonal, Santa Cruz biotechnology), anti-CTIP2 (mouse monoclonal, Abcam), anti-Nkx2.1 (rabbit polyclonal, Santa Cruz biotechnology), anti-Sox2 (human-derived mouse/rat/human monoclonal, R&D Systems).

Techniques: Expressing

Journal: Neuron

Article Title: Radial glial lineage progression and differential intermediate progenitor amplification underlie striatal compartments and circuit organization

doi: 10.1016/j.neuron.2018.06.021

Figure Lengend Snippet: (A) Fluorescent in situ hybridization (FISH) signal from a branched-chain DNA (bDNA) probe targeting the tdTomato mRNA (red) in P28 SPNs following E10.5 TM in Ascl1-CreER;Ai14 mice matches the signature pattern (arrows) of striosomal cells along the lateral border of striatum. Blue: Nissl. Scale bars: 300 µm in left panel, 100 µm in right panel.

Article Snippet: Sections were blocked in 10% normal goat serum and 0.1% triton-X100 in 1XPBS, then incubated in primary antibodies (diluted in blocking solution) at 4°C overnight: anti-RFP (rabbit polyclonal antibody, Rockland Pharmaceuticals), anti-MOR1 (rabbit polyclonal, immunostar), anti-Calbindin 28K (rabbit polyclonal, Millipore), anti-CDG1 (rabbit polyclonal, Crittenden et al., 2009 ), anti-Tyrosine Hydroxylase (rabbit polyclonal, Millipore), anti-Ki67 (rabbit polyclonal, Vector), anti-Ascl1/Mash1 (mouse monoclonal, BD biosciences), anti-BrdU (rat monoclonal Accurate Chemical), anti-TuJ1 (mouse monoclonal, Covance), anti-Parvalbumin (mouse monoclonal, Sigma), anti-Somatostatin (rabbit polyclonal, Millipore), anti-ChAT (rabbit polyclonal, Santa Cruz biotechnology), anti-CTIP2 (mouse monoclonal, Abcam), anti-Nkx2.1 (rabbit polyclonal, Santa Cruz biotechnology), anti-Sox2 (human-derived mouse/rat/human monoclonal, R&D Systems).

Techniques: In Situ Hybridization

Journal: Neurobiology of disease

Article Title: N-butyl-deoxynojirimycin delays motor deficits, cerebellar microgliosis, and Purkinje cell loss in a mouse model of mucolipidosis type IV

doi: 10.1016/j.nbd.2017.06.003

Figure Lengend Snippet: A) Immunofluorescent detection of GM2 in neocortical cerebral tissue. Max intensity projection of equivalent number of slices for each image. B,C) Lipid analysis via mass spectrometry of the major isoform (i.e. most prevalent) of GM2 in both cerebrum (B) and cerebellum (C). n=4 per group. Differences detected in both cerebrum and cerebellum in genotype by ANOVA and results of post hoc pairwise comparisons noted. All pairwise comparisons were made using Tukey’s multiple comparisons tests when appropriate, with significant differences between WT saline - Mcoln1−/− saline, Mcoln1−/− saline - Mcoln1−/− miglustat, WT saline - Mcoln1−/− miglustat, and WT saline - WT miglustat denoted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were then incubated with primary antibody in diluent solution (5% normal goat serum, 0.02% saponin, 1× PBS) overnight at 4° C. Primary antibody(ies) included anti-GM2 (1:50, mouse IgM, cell culture supernatant produced in-house by Dr. K. Dobrenis using the 10–11 hybridoma line from Progenics Pharmaceuticals, Tarrytown, NY USA), anti-CD68 (1:500, rat monoclonal antibody, AbD Serotec MCA1957), anti-GFAP (1:1000, rabbit polyclonal antibody, Sigma G9269), and anti-Calbindin-D 28K (1:500, mouse monoclonal antibody, Sigma C9848).

Techniques: Mass Spectrometry

Journal: Neurobiology of disease

Article Title: N-butyl-deoxynojirimycin delays motor deficits, cerebellar microgliosis, and Purkinje cell loss in a mouse model of mucolipidosis type IV

doi: 10.1016/j.nbd.2017.06.003

Figure Lengend Snippet: A) Schematic of GSL synthesis pathway and abbreviations used. Purported point of action of miglustat denoted in red. B–D) Total levels detected for each lipid listed in cerebellum, including those enriched in white matter (B), complex gangliosides (C), and ceramide & globo-series glycolipids (D). E–I) Major (ie, most prevalent) isoforms detected are plotted for each lipid (complete isoform analysis plotted in Supp. Fig.3–5.), including ceramides (E, ratio of ceramides C18:0 to C24:1 are plotted in F), CMH (G), sulfatides (H), and GM1 (I). n=4 per group. Differences were detected by two-way ANOVA for genotype in total CMH, total CDH, total sulfatides, total GM2, C24:1 CMH, and C24:1 sulfatide; for treatment in the ratio of C18:0/C24:1 ceramide; and for both genotype and treatment in total GM1 and C18:0 GM1. Where appropriate, post hoc pairwise comparisons were made with significant differences detected between WT saline - Mcoln1−/− saline, Mcoln1−/− saline - Mcoln1−/− miglustat, WT saline - Mcoln1−/− miglustat, and WT saline - WT miglustat denoted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were then incubated with primary antibody in diluent solution (5% normal goat serum, 0.02% saponin, 1× PBS) overnight at 4° C. Primary antibody(ies) included anti-GM2 (1:50, mouse IgM, cell culture supernatant produced in-house by Dr. K. Dobrenis using the 10–11 hybridoma line from Progenics Pharmaceuticals, Tarrytown, NY USA), anti-CD68 (1:500, rat monoclonal antibody, AbD Serotec MCA1957), anti-GFAP (1:1000, rabbit polyclonal antibody, Sigma G9269), and anti-Calbindin-D 28K (1:500, mouse monoclonal antibody, Sigma C9848).

Techniques:

Journal: Neurobiology of disease

Article Title: N-butyl-deoxynojirimycin delays motor deficits, cerebellar microgliosis, and Purkinje cell loss in a mouse model of mucolipidosis type IV

doi: 10.1016/j.nbd.2017.06.003

Figure Lengend Snippet: A–C) Total levels of lipids enriched in cerebral white matter, complex gangliosides, and ceramide & globo-series glycolipids are plotted as in Fig. 4. D–H) Major isoforms detected are plotted for each lipid as in Fig. 4 (complete isoform analysis plotted in Supp. Fig. 3–5), including ceramides (D, ratio of ceramides C18:0 to C24:1 are plotted in E), CMH (F), sulfatides (G), and GM1 (H). n=4 per group. Differences were detected by two-way ANOVA for genotype in total CMH, total CDH, total sulfatides, total GM2, total GM3, C18:0 Cer, C24:1 Cer, C24:1 CMH, and C24:1 sulfatide; for treatment in total GM1, total Gb3Cer, total Gb4Cer, the ratio of C18:0/C24:1 ceramide, and C18:0 GM1. Pairwise comparisons were made when appropriate, with significant differences between WT saline - Mcoln1−/− saline, Mcoln1−/− saline - Mcoln1−/− miglustat, WT saline - Mcoln1−/− miglustat, and WT saline - WT miglustat denoted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were then incubated with primary antibody in diluent solution (5% normal goat serum, 0.02% saponin, 1× PBS) overnight at 4° C. Primary antibody(ies) included anti-GM2 (1:50, mouse IgM, cell culture supernatant produced in-house by Dr. K. Dobrenis using the 10–11 hybridoma line from Progenics Pharmaceuticals, Tarrytown, NY USA), anti-CD68 (1:500, rat monoclonal antibody, AbD Serotec MCA1957), anti-GFAP (1:1000, rabbit polyclonal antibody, Sigma G9269), and anti-Calbindin-D 28K (1:500, mouse monoclonal antibody, Sigma C9848).

Techniques: