Journal: Frontiers in Molecular Neuroscience

Article Title: Cloning and Phylogenetic Analysis of NMDA Receptor Subunits NR1, NR2A and NR2B in Xenopus laevis Tadpoles

doi: 10.3389/neuro.02.004.2009

Figure Lengend Snippet: Detection of NMDAR subunits and splice variants in X. laevis tadpole brain . (A) Immunoblots for NR1, NR2A and NR2B were done on whole brain lysates of stage 47/48 tadpoles (Xen) with antibodies generated against homologous rat proteins. Immunoreactive bands of about the same size as in rat whole brain lysate (Rat) were found for all three subunits. (B) Western blots of whole cell lysates of HEK293 cells transfected with X. laevis the NR1-4a/b splice variant or NR2A cDNA, and their mock-transfected controls. (C) Cartoon of NR1 splice variants and the alternatively spliced exons (N1, C1, C2) and C-terminal ends (C2′) that they contain. (D) Cross-reactive bands for exons N1 and C1, as well as alternative C-terminal end C2′ in X. laevis were detected with antibodies to rat homologs by Western blot on whole brain extracts from stage 47/48 tadpoles and rat. No cross-reactive band was detected for C2. (E,F) C-terminal ends of NR1 mRNAs were amplified by RT-PCR and PCR with primer pairs 5′ to the spliced region and in the 3′UTR. (E) Twenty-six out of 29 nucleotide sequences contained only C2′ (Xen_NR1_C2′). They aligned perfectly to the published X. laevis NR1 sequence (Xen_NR1_X94156). (F) Two out of 29 nucleotide sequences contained C1 and C2′ (Xen_NR1_C1_C2′), indicating that NR1-3a/b also exists in X. laevis . The alignment to rat NR1-3a (Rat_NR1-3a) shows a very high degree of sequence conservation. Identical residues are marked with (*).

Article Snippet: An equal amount of protein per lane was separated by 8% SDS-PAGE and transferred onto nitrocellulose membrane that were probed with primary antibodies at 1:100–500: α-NR1, α-NR2B (both BD PharMingen), α-NR2A (crude rabbit serum JH 1817, gift from the Huganir Lab), α-NR1-C1 (crude rabbit serum JH 2079, gift from the Huganir Lab), α-NR1-N1, α-NR1-C2 and α-NR1-C2’ (all three Novus Biologicals) and incubated with the HRP-conjugated α-goat or α-mouse secondary antibody (Bio-Rad, 1:3000).

Techniques: Western Blot, Generated, Transfection, Variant Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Human Molecular Genetics

Article Title: Ataxia is the major neuropathological finding in arylsulfatase G-deficient mice: similarities and dissimilarities to Sanfilippo disease (mucopolysaccharidosis type III)

doi: 10.1093/hmg/ddu603

Figure Lengend Snippet: Expression of ARSG in the murine central nervous system. (A) Immunoblot analysis of different brain areas (Cb = cerebellum, Hc = hippocampus, Cx = cortex, Ob = olfactory bulb, Bs = brain stem, Th = thalamus, Sc = spinal cord) shows highest expression of ARSG in the spinal cord and the brain stem and lowest expression in the olfactory bulb. Homogenates from Arsg KO mice (−/−) served as controls for specificity of the antibody. ARSG-specific bands are labelled with arrows. A non-specific band also present in KO mice is labelled with an asterisk. (B) ARSG was specifically detected with a polyclonal antibody in wild-type sections with particularly high expression in the anterior horn of the spinal cord and the inferior colliculus. Absence of specific DAB staining reveals specificity of the antibody. Scale bars = 200 µm (C) Double immunofluorescence shows co-localization of ARSG and LAMP1 with additional cytoplasmic staining for ARSG in cells of the spinal cord. ARSG staining is absent in the spinal cord of KO mice. Scale bars = 15 µm. (D) Double immunofluorescence staining of ARSG with markers for different CNS cell types reveals modest expression in microglia (Iba1+), highest expression in perivascular macrophages (CD68+) but no expression in astrocytes (GFAP+). Oligodendrocytes (Olig-2+ and CNPase+) reveal extensive expression of ARSG, while neurons (NeuN+) reveal only subtle staining. Endothelial (Pecam-1+) cells lack ARSG staining. All pictures from the inferior colliculus, except CNPase (granular layer of the cerebellum). Scale bars = 15 µm. (E) Semi-quantitative grading of the ARSG expression in wild-type cells and comparison with lysosomal storage phenotype in Arsg KO mouse cells reveal that highest expression in perivascular cells is paralleled with the highest vacuolization. However, oligodendrocytes with high expression do not reveal signs of lysosomal storage. (1)not determined due to non-specific antibody reactivity in PCs; (2)lysosomes containing phagocytosed debris were considered to be unrelated to the primary enzyme defect.

Article Snippet: Antibodies and chemicals Primary antibodies used in this study: Calbindin (Sigma Aldrich), GFAP (Sigma Aldrich), CNPase, ARSG (R&D Systems), NeuN (Millipore), LAMP1 (clone 1D4B, Developmental Studies Hybridoma Bank), CD68 (AbD Serotec), Iba1 (Genetex), MBP (Millipore), Gapdh (Santa Cruz), p62 (Enzo Life Sciences), ubiquitin (Dako), LC3 (Novus Biologicals), subunit c of mitochondrial ATP synthase (SCMAS) (described in 10 ) and GM2 (mab10–11, Progenics Pharmaceuticals), GM3 (DH2, mouse IgG3, GlycoTech Corp.).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Double Immunofluorescence Staining, Comparison

Journal: Human Molecular Genetics

Article Title: Ataxia is the major neuropathological finding in arylsulfatase G-deficient mice: similarities and dissimilarities to Sanfilippo disease (mucopolysaccharidosis type III)

doi: 10.1093/hmg/ddu603

Figure Lengend Snippet: Accumulation of aggregated proteins in the cerebellum. (A) Western blots of detergent-soluble fractions of the cerebellum show no increase in LC3-II of 12-month-old and 24-month-old Arsg KO mice, but slight increase of p62 and phospho-p62 in detergent-insoluble extracts. (B) Electron micrograph of a PC axon reveals increased amounts of autophagosomes that are, however, rarely seen, whereas in the molecular layer of the cerebellum large filamentous structures (C) of up to 10 µm can be regularly found. Scale bars: b = 1 µm; c = 5 µm; inset = 1 µm. (D) Immunohistochemical staining of p62 and ubiquitin of the cerebellum reveals large aggregates with a diameter of up to 6 µm in the molecular layer of the cerebellum, which are absent in wild-type mice (age 22 months). Scale bar = 25 µm. (E) Co-immunofluorescence of p62 (red) and ubiquitin (green) reveals extensive co-localization. Scale bar = 10 µm. (F) In contrast, p62 aggregates do not co-localize at all or only weakly with Lamp1 (upper panel). They are often found in very close proximity to Lamp1-positive lysosomes (lower panels) and higher magnification reveals p62 decorating lysosomal membranes (arrow heads). Scale bars = 5 µm. (G) Co-localization with autofluorescent lipofuscin indicates similarly p62 decorating lipofuscin aggregates. Scale bar = 5 µm. (H) Western blot of detergent-insoluble cerebellar extracts from 12-month-old and 24-month-old animals for ubiquitin reveals increased levels of several ubiquitinated protein species of ∼25–35 kDa (labeled with a bracket).

Article Snippet: Antibodies and chemicals Primary antibodies used in this study: Calbindin (Sigma Aldrich), GFAP (Sigma Aldrich), CNPase, ARSG (R&D Systems), NeuN (Millipore), LAMP1 (clone 1D4B, Developmental Studies Hybridoma Bank), CD68 (AbD Serotec), Iba1 (Genetex), MBP (Millipore), Gapdh (Santa Cruz), p62 (Enzo Life Sciences), ubiquitin (Dako), LC3 (Novus Biologicals), subunit c of mitochondrial ATP synthase (SCMAS) (described in 10 ) and GM2 (mab10–11, Progenics Pharmaceuticals), GM3 (DH2, mouse IgG3, GlycoTech Corp.).

Techniques: Western Blot, Immunohistochemical staining, Staining, Ubiquitin Proteomics, Immunofluorescence, Labeling

Journal: Human Molecular Genetics

Article Title: Ataxia is the major neuropathological finding in arylsulfatase G-deficient mice: similarities and dissimilarities to Sanfilippo disease (mucopolysaccharidosis type III)

doi: 10.1093/hmg/ddu603

Figure Lengend Snippet: Secondary storage of SCMAS in the cerebral cortex but not the cerebellum. (A) Immunofluorescence staining of SCMAS in 22-month-old wild-type and Arsg KO mice reveals clearly increased amounts of SCMAS in layer V of the cerebral cortex, but comparable levels of SCMAS in the cerebellum of wild-type and Arsg KO mice. Scale bars = 100 µm (upper panel); 20 µm (lower panel). (B) SCMAS partially, but not completely, co-localizes with LAMP1 in cortical neurons of Arsg KO mouse as depicted by higher magnification. Scale bar = 10 µm.

Article Snippet: Antibodies and chemicals Primary antibodies used in this study: Calbindin (Sigma Aldrich), GFAP (Sigma Aldrich), CNPase, ARSG (R&D Systems), NeuN (Millipore), LAMP1 (clone 1D4B, Developmental Studies Hybridoma Bank), CD68 (AbD Serotec), Iba1 (Genetex), MBP (Millipore), Gapdh (Santa Cruz), p62 (Enzo Life Sciences), ubiquitin (Dako), LC3 (Novus Biologicals), subunit c of mitochondrial ATP synthase (SCMAS) (described in 10 ) and GM2 (mab10–11, Progenics Pharmaceuticals), GM3 (DH2, mouse IgG3, GlycoTech Corp.).

Techniques: Immunofluorescence, Staining