Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures
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{"title"=>"Early increase and late decrease of Purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures", "type"=>"journal", "authors"=>[{"first_name"=>"Jody L.", "last_name"=>"Campeau", "scopus_author_id"=>"55331470500"}, {"first_name"=>"Gengshu", "last_name"=>"Wu", "scopus_author_id"=>"57199960768"}, {"first_name"=>"John R.", "last_name"=>"Bell", "scopus_author_id"=>"55985153200"}, {"first_name"=>"Jay", "last_name"=>"Rasmussen", "scopus_author_id"=>"36768059600"}, {"first_name"=>"Valerie L.", "last_name"=>"Sim", "scopus_author_id"=>"8946806200"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84891612408", "pui"=>"372039755", "doi"=>"10.1371/journal.pone.0081776", "isbn"=>"1932-6203; 1932-6203", "sgr"=>"84891612408", "pmid"=>"24312586"}, "id"=>"25c393e5-8a29-378e-8f02-1b184ac1ae66", "abstract"=>"Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.", "link"=>"http://www.mendeley.com/research/early-increase-late-decrease-purkinje-cell-dendritic-spine-density-prioninfected-organotypic-mouse-c", "reader_count"=>18, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>6, "Student > Master"=>4, "Student > Bachelor"=>1, "Professor"=>3}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>6, "Student > Master"=>4, "Student > Bachelor"=>1, "Professor"=>3}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>10, "Medicine and Dentistry"=>2, "Neuroscience"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>10}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}}, "reader_count_by_country"=>{"United Kingdom"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1299839"], "description"=>"<p>A) The typical banding pattern of RML-infected brain homogenate (RML BH) inoculum after proteinase K (PK) digestion is shown. 10, 5, and 2 µg total protein were loaded. B) RML-infected POSCA slices were homogenized in pairs in RIPA buffer, then 30 µg total protein was treated with 20 µg/mL PK for one hour (+). Undigested slice culture homogenates (−) were loaded at 10x lower concentration for comparison. PrP<sup>Sc</sup> is distinguished by its truncated state after PK digestion, seen as lower molecular weight bands when compared with PrP<sup>C</sup>. PrP<sup>Sc</sup> can also been seen to increase in amount with increasing culture time (days 7 through 42 are shown). C) RML-treated POSCA slices from <i>Prnp<sup>0/0</sup></i> mice did not show PrP<sup>Sc</sup> at any time point. Day 45 is shown.</p>", "links"=>[], "tags"=>["cerebellar", "cultures", "infected", "rml"], "article_id"=>865796, "categories"=>["Biological Sciences"], "users"=>["Jody L. Campeau", "Gengshu Wu", "John R. Bell", "Jay Rasmussen", "Valerie L. Sim"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0081776.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Immunoblots_of_PrP_Sc_in_cerebellar_slice_cultures_infected_with_RML_strain_/865796", "title"=>"Immunoblots of PrP<sup>Sc</sup> in cerebellar slice cultures infected with RML strain.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-02 02:50:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1299840"], "description"=>"<p>A,B) Representative Z-stacked image of a Purkinje cell as analyzed with Imaris 7.1.1 software, after deconvolution with ImageQuant X. B) Inset of A showing dendritic spine analysis; spines identified in blue. Nuclei are stained with Hoechst (blue), dead cells are stained with propidium iodide (red) and Purkinje cells are labelled with calbindin/Alexafluor488 (green). Scale bar 10 µm. C) Average spine density from uninfected cerebellar cultures from C57Bl6 mice at weekly time points. Errors bars represent one standard error of the mean. n = 9 except day 14 where n = 3.</p>", "links"=>[], "tags"=>["uninfected", "cerebellar", "cultures", "c57bl6"], "article_id"=>865797, "categories"=>["Biological Sciences"], "users"=>["Jody L. Campeau", "Gengshu Wu", "John R. Bell", "Jay Rasmussen", "Valerie L. Sim"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0081776.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Purkinje_cell_spine_density_analysis_stability_in_uninfected_cerebellar_slice_cultures_from_C57Bl6_mice_/865797", "title"=>"Purkinje cell spine density analysis; stability in uninfected cerebellar slice cultures from C57Bl6 mice.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-02 02:50:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1299841"], "description"=>"<p>Low resolution images (2X) of uninfected (A) and infected (B) cerebellar slices at day 42, demonstrating preserved cerebellar architecture and Purkinje cells. Imaged on an InCell Analyzer. Nuclei are stained with Hoechst (blue) and Purkinje cells are labelled with Calbindin/Alexafluor488 (green).</p>", "links"=>[], "tags"=>["42", "days"], "article_id"=>865798, "categories"=>["Biological Sciences"], "users"=>["Jody L. Campeau", "Gengshu Wu", "John R. Bell", "Jay Rasmussen", "Valerie L. Sim"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0081776.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cerebellar_slice_architecture_after_42_days_of_culture_/865798", "title"=>"Cerebellar slice architecture after 42 days of culture.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-02 02:50:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1299848"], "description"=>"<p>A) Average Purkinje cell spine densities at weekly time points. B–G) Representative images from uninfected (B–D) and RML-infected (E–G) cultures at day 69. Nuclei are stained with Hoechst (blue), dead cells are stained with propidium iodide (red) and Purkinje cells are labelled with Calbindin/Alexafluor488 (green). Scale bar 10 µm. Insets below each panel are magnified by a factor of three to better visualize dendritic spines. Images of fixed samples were taken on a Zeiss LSM 700, 63x oil objective, deconvolved with ImageQuant X and analyzed using Imaris 7.1.1 software. The average n was 23 per time point for infected (range 12–41), 14 for uninfected (range 6–22). At day 69, n = 17 for infected, n = 21 for uninfected. p-values: * <0.05; ** <0.01; *** <0.001.</p>", "links"=>[], "tags"=>["dendritic", "densities", "rml-infected", "uninfected", "cerebellar"], "article_id"=>865805, "categories"=>["Biological Sciences"], "users"=>["Jody L. Campeau", "Gengshu Wu", "John R. Bell", "Jay Rasmussen", "Valerie L. Sim"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0081776.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Purkinje_cell_dendritic_spine_densities_in_RML_infected_and_uninfected_cerebellar_slice_cultures_/865805", "title"=>"Purkinje cell dendritic spine densities in RML-infected and uninfected cerebellar slice cultures.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-02 02:50:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1299849"], "description"=>"<p>A) Representative immunoblot of PrP<sup>Sc</sup> in infected slice cultures before (−) and after (+) proteinase K (PK) digestion. IPC1 antibody was the primary antibody. 4 of 5 experiments first detected PrP<sup>Sc</sup> at day 21, 1 was detected by day 17. Undigested samples were diluted by 10x before loading. B) 2–3 slices were collected at days 10 through 27, normalized for total protein by BCA assay, and probed for post-synaptic density 95 (PSD95), pre-synaptic synaptophysin (synapt.), GAPDH (protein loading control), GFAP (astrocyte marker) and NeuN (neuronal marker). C) Levels of PSD95, normalized to GAPDH, in infected and uninfected cultures at weekly time points. Differences were not statistically significant. D) Levels of synaptophysin, normalized to GAPDH, in infected and uninfected cultures at weekly time points. Differences were not statistically significant. E) Ratios of GAPDH-normalized synaptophysin/PSD95 are shown for infected and uninfected cultures at weekly time points. Each data point represents a single ratio from averages from all experiments.</p>", "links"=>[], "tags"=>["rml-infected", "uninfected", "cerebellar", "cultures", "tga20", "mice"], "article_id"=>865806, "categories"=>["Biological Sciences"], "users"=>["Jody L. Campeau", "Gengshu Wu", "John R. Bell", "Jay Rasmussen", "Valerie L. Sim"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0081776.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Immunoblots_of_RML_infected_and_uninfected_cerebellar_slice_cultures_from_tga20_mice_at_early_time_points_/865806", "title"=>"Immunoblots of RML-infected and uninfected cerebellar slice cultures from tga20 mice at early time points.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-02 02:50:24"}

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Relative Metric

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