Journal: Science Advances

Article Title: The yeast prion protein Sup35 initiates α-synuclein pathology in mouse models of Parkinson’s disease

doi: 10.1126/sciadv.adj1092

Figure Lengend Snippet: ( A ) Representative images of α-syn pathology in different brain regions of control mice, mice with S. cerevisiae nasal infection, and mice with S. cerevisiae nasal infection together with fluconazole treatment ( n = 4 mice per group). Scale bar, 20 μm. PBS, phosphate-buffered saline; IHC, immunohistochemistry. ( B and C ) Representative TH immunostaining of dopaminergic neurons in the right SNpc region. The bar graph shows the quantification of the TH-positive neurons ( n = 5 mice per group). Scale bar, 100 μm. ( D ) Representative images of TH immunostaining of the striatum. The bar graph shows the optical density analysis ( n = 4 mice per group). Scale bar, 200 μm. ( E and F ) Western blots showing the levels of TH and DAT in the striatum. The bar graph shows the quantification of TH and DAT ( n = 4 mice per group). Data are presented as means ± SEM . P values were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons [(D) and (F)]. ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., not significant; GAPDH, glyceraldehyde phosphate dehydrogenase.

Article Snippet: The following primary antibodies were used in this study: GFP (Proteintech, 66002-1-Ig), Sup35 (Abnoca, PAB15578), glyceraldehyde phosphate dehydrogenase (Proteintech, 60004-1-Ig), phospho–α-Syn (Ser 129 ) (Biolegend, MMS-5091), phospho–α-Syn (Ser 129 ) (Cell Signaling Technology, 23706), synaptophysin (Proteintech, 17785-1-AP), PSD-95 (20665-1-AP), synapsin I (Proteintech, 20258-1-AP), α-syn (syn211, Invitrogen, MA5-12272), glutathione S -transferase (66001-2-Ig), NeuN (Millipore, MAB377), TH (Millipore, AB152), DAT (Abcam, ab184451), ubiquitin (Cell Signaling Technology, 3936S), p62 (Abcam, 56416), Microtuble-associated protein 2 (MAP2) (Proteintech, 17490-1-AP and Invitrogen, 13-1500), tau (Thermo, MA5-12808), Bcl2 (Cell Signaling Technology, 15071), Bax (Abcam, ab32503), Cox IV (Abcam, ab16056), Alexa Fluor 488-Goat anti-Mouse (Invitrogen, A11001), Alexa Fluor 594-Goat anti-Mouse (Invitrogen, A11005), Alexa Fluor 488-Goat anti-Rabbit (Invitrogen, A11034), and Alexa Fluor 594-Goat anti-Rabbit (Invitrogen, A11012).

Techniques: Control, Infection, Saline, Immunohistochemistry, Immunostaining, Western Blot

Journal: Science Advances

Article Title: The yeast prion protein Sup35 initiates α-synuclein pathology in mouse models of Parkinson’s disease

doi: 10.1126/sciadv.adj1092

Figure Lengend Snippet: ( A ) Colocalization of pS129 and Sup35 in the SN of brain slices from patients with PD. The bar graph is quantification of the colocalization between Sup35 and pS129 ( n = 8 cases per group). DAPI, 4′,6-diamidino-2-phenylindole. ( B and C ) His pull-down assay (B) and label transfer protein interaction analysis reveals the interaction between α-syn and Sup35 (C). ( D and E ) The kinetics of α-syn [1.5 mg/ml (D) and 0.75 mg/ml (E)] fibrillization in the presence or absence of Sup35 and mutant Sup35 (0.05 mg/ml) as monitored by ThT fluorescence. AFU, arbitrary fluorescence unit. ( F ) Sup35 (green) coaggregates with α-syn (red) in HEK293 cells. ( G ) α-Syn aggregation and pS129 signals induced by Sup35 PFFs in α-syn–HEK293 cells. The bar graph shows the percentage of cells with pS129 ( n = 10 visual fields per group from four independent experiments). ( H ) α-Syn aggregation at 6, 10, and 15 days after transduction with Sup35 PFFs and mutant Sup35. ( I ) Sedimentation analysis showing that Sup35 PFFs induced the formation of high–molecular weight α-syn species in neurons. T, total fraction, S, supernatant, P, pellet. ( J ) Biotin-labeled Sup35 PFFs (red) were surrounded by pS129 signals (green). ( K ) Immunofluorescence showing the expression of synapsin I in neurons. ( L ) Representative images and quantification of DiI staining of dendritic spines in neurons treated with phosphate-buffered saline (PBS) or Sup35 PFFs for 15 days ( n = 15 fields per group). Data are presented as means ± SEM . P values were determined by Mann-Whitney test [(A) and (L)] or one-way ANOVA followed by Tukey’s multiple comparisons (G). ** P < 0.01, **** P < 0.0001. Scale bar, 20 μm.

Article Snippet: The following primary antibodies were used in this study: GFP (Proteintech, 66002-1-Ig), Sup35 (Abnoca, PAB15578), glyceraldehyde phosphate dehydrogenase (Proteintech, 60004-1-Ig), phospho–α-Syn (Ser 129 ) (Biolegend, MMS-5091), phospho–α-Syn (Ser 129 ) (Cell Signaling Technology, 23706), synaptophysin (Proteintech, 17785-1-AP), PSD-95 (20665-1-AP), synapsin I (Proteintech, 20258-1-AP), α-syn (syn211, Invitrogen, MA5-12272), glutathione S -transferase (66001-2-Ig), NeuN (Millipore, MAB377), TH (Millipore, AB152), DAT (Abcam, ab184451), ubiquitin (Cell Signaling Technology, 3936S), p62 (Abcam, 56416), Microtuble-associated protein 2 (MAP2) (Proteintech, 17490-1-AP and Invitrogen, 13-1500), tau (Thermo, MA5-12808), Bcl2 (Cell Signaling Technology, 15071), Bax (Abcam, ab32503), Cox IV (Abcam, ab16056), Alexa Fluor 488-Goat anti-Mouse (Invitrogen, A11001), Alexa Fluor 594-Goat anti-Mouse (Invitrogen, A11005), Alexa Fluor 488-Goat anti-Rabbit (Invitrogen, A11034), and Alexa Fluor 594-Goat anti-Rabbit (Invitrogen, A11012).

Techniques: Pull Down Assay, Mutagenesis, Fluorescence, Transduction, Sedimentation, High Molecular Weight, Labeling, Immunofluorescence, Expressing, Staining, Saline, MANN-WHITNEY

Journal: Science Advances

Article Title: The yeast prion protein Sup35 initiates α-synuclein pathology in mouse models of Parkinson’s disease

doi: 10.1126/sciadv.adj1092

Figure Lengend Snippet: ( A ) Representative images of α-syn pathology in mice injected with PBS, Sup35 PFFs, and Curli PFFs at 1, 4, and 7 mpi. Scale bar, 20 μm. ( B ) Heatmaps of pS129 pathological scores in mice injected with Sup35 PFFs. The severity of α-syn pathology is reflected by the number and size of the aggregates. The blue dot in the stratum represents the injection site ( n = 5 to 6 mice per group).

Article Snippet: The following primary antibodies were used in this study: GFP (Proteintech, 66002-1-Ig), Sup35 (Abnoca, PAB15578), glyceraldehyde phosphate dehydrogenase (Proteintech, 60004-1-Ig), phospho–α-Syn (Ser 129 ) (Biolegend, MMS-5091), phospho–α-Syn (Ser 129 ) (Cell Signaling Technology, 23706), synaptophysin (Proteintech, 17785-1-AP), PSD-95 (20665-1-AP), synapsin I (Proteintech, 20258-1-AP), α-syn (syn211, Invitrogen, MA5-12272), glutathione S -transferase (66001-2-Ig), NeuN (Millipore, MAB377), TH (Millipore, AB152), DAT (Abcam, ab184451), ubiquitin (Cell Signaling Technology, 3936S), p62 (Abcam, 56416), Microtuble-associated protein 2 (MAP2) (Proteintech, 17490-1-AP and Invitrogen, 13-1500), tau (Thermo, MA5-12808), Bcl2 (Cell Signaling Technology, 15071), Bax (Abcam, ab32503), Cox IV (Abcam, ab16056), Alexa Fluor 488-Goat anti-Mouse (Invitrogen, A11001), Alexa Fluor 594-Goat anti-Mouse (Invitrogen, A11005), Alexa Fluor 488-Goat anti-Rabbit (Invitrogen, A11034), and Alexa Fluor 594-Goat anti-Rabbit (Invitrogen, A11012).

Techniques: Injection

Journal: Science Advances

Article Title: The yeast prion protein Sup35 initiates α-synuclein pathology in mouse models of Parkinson’s disease

doi: 10.1126/sciadv.adj1092

Figure Lengend Snippet: ( A ) Representative images of α-syn pathology in different brain regions of mice injected with PBS, α-syn PFFs, or S-α-syn PFFs at 1, 3, and 7 mpi ( n = 4 mice per group). Scale bar, 20 μm. ( B and C ) Sequential extraction experiments showing the presence of high–molecular weight α-syn species in the ipsilateral striatum and SN of mice injected with PBS, α-syn PFFs, and S-α-syn PFFs for 7 months ( n = 4 mice per group). Data are presented as means ± SEM . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons [(B) and (C)]. *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The following primary antibodies were used in this study: GFP (Proteintech, 66002-1-Ig), Sup35 (Abnoca, PAB15578), glyceraldehyde phosphate dehydrogenase (Proteintech, 60004-1-Ig), phospho–α-Syn (Ser 129 ) (Biolegend, MMS-5091), phospho–α-Syn (Ser 129 ) (Cell Signaling Technology, 23706), synaptophysin (Proteintech, 17785-1-AP), PSD-95 (20665-1-AP), synapsin I (Proteintech, 20258-1-AP), α-syn (syn211, Invitrogen, MA5-12272), glutathione S -transferase (66001-2-Ig), NeuN (Millipore, MAB377), TH (Millipore, AB152), DAT (Abcam, ab184451), ubiquitin (Cell Signaling Technology, 3936S), p62 (Abcam, 56416), Microtuble-associated protein 2 (MAP2) (Proteintech, 17490-1-AP and Invitrogen, 13-1500), tau (Thermo, MA5-12808), Bcl2 (Cell Signaling Technology, 15071), Bax (Abcam, ab32503), Cox IV (Abcam, ab16056), Alexa Fluor 488-Goat anti-Mouse (Invitrogen, A11001), Alexa Fluor 594-Goat anti-Mouse (Invitrogen, A11005), Alexa Fluor 488-Goat anti-Rabbit (Invitrogen, A11034), and Alexa Fluor 594-Goat anti-Rabbit (Invitrogen, A11012).

Techniques: Injection, Extraction, High Molecular Weight

Journal: Science Advances

Article Title: The yeast prion protein Sup35 initiates α-synuclein pathology in mouse models of Parkinson’s disease

doi: 10.1126/sciadv.adj1092

Figure Lengend Snippet: ( A ) Double-labeling immunofluorescence of pS129 (green) and TH (red) in the SNpc of mice injected with PBS, α-syn PFFs, and S-α-syn PFFs ( n = 4 mice per group). Scale bar, 20 μm. ( B ) Representative TH immunostaining of dopaminergic neurons in the SNpc of mice injected with PBS, α-syn PFFs or S-α-syn PFFs at 7 mpi. The bar graph shows the quantification of the TH-positive neurons ( n = 5 mice per group). Scale bar, 100 μm. ( C ) TH immunostaining of the striatum ( n = 4 mice per group). Scale bar, 200 μm. ( D ) Western blots showing the levels of striatal TH and DAT ( n = 4 mice per group). ( E ) HPLC analysis of striatal DA, DOPAC, and HVA ( n = 5 mice per group). ( F ) Representative images of the tail suspension test. ( G to L ) Rotarod test, grid test, pole test, balance beam test, and footprint test were performed at 7 mpi in mice injected with PBS, α-syn PFFs, and S-α-syn PFFs ( n = 10 mice per group). ( M ) Western blots showing the levels of Cox IV, Bcl2, and Bax in the striatum. The bar graph shows the quantification of the levels of Cox IV, Bcl2, and Bax ( n = 4 mice per group). ( N ) Representative image of striatal mitochondrial morphology. The bar graph shows the quantification of the percentage of damaged mitochondria. More than 200 mitochondria were analyzed from each mouse sample ( n = 4 mice in each group). Scale bar, 1 μm in the upper panel and 200 nm in the lower panel. The PBS control group was shared in Figs. 4 and . Data are presented as means ± SEM . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons [(B) to (E) and (G) to (N)]. *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The following primary antibodies were used in this study: GFP (Proteintech, 66002-1-Ig), Sup35 (Abnoca, PAB15578), glyceraldehyde phosphate dehydrogenase (Proteintech, 60004-1-Ig), phospho–α-Syn (Ser 129 ) (Biolegend, MMS-5091), phospho–α-Syn (Ser 129 ) (Cell Signaling Technology, 23706), synaptophysin (Proteintech, 17785-1-AP), PSD-95 (20665-1-AP), synapsin I (Proteintech, 20258-1-AP), α-syn (syn211, Invitrogen, MA5-12272), glutathione S -transferase (66001-2-Ig), NeuN (Millipore, MAB377), TH (Millipore, AB152), DAT (Abcam, ab184451), ubiquitin (Cell Signaling Technology, 3936S), p62 (Abcam, 56416), Microtuble-associated protein 2 (MAP2) (Proteintech, 17490-1-AP and Invitrogen, 13-1500), tau (Thermo, MA5-12808), Bcl2 (Cell Signaling Technology, 15071), Bax (Abcam, ab32503), Cox IV (Abcam, ab16056), Alexa Fluor 488-Goat anti-Mouse (Invitrogen, A11001), Alexa Fluor 594-Goat anti-Mouse (Invitrogen, A11005), Alexa Fluor 488-Goat anti-Rabbit (Invitrogen, A11034), and Alexa Fluor 594-Goat anti-Rabbit (Invitrogen, A11012).

Techniques: Labeling, Immunofluorescence, Injection, Immunostaining, Western Blot, Suspension, Control

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Antibodies used for immunofluorescence, immunohistochemistry, and Western blot assays

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Immunofluorescence, Immunohistochemistry, Western Blot, Ubiquitin Proteomics

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Challenge with α-synuclein fibrils causes disease in bigenic Tg(M83 +/− : Gfap -luc +/− ) mice. (A) Negatively stained electron micrograph of sonicated recombinant human α-synuclein fibrils that were injected into mice. Aggregated samples were stained with uranyl acetate. Multiple clusters of short, rod-shaped aggregates are visible. Bar = 100 nm. (B) Kaplan-Meier survival curves show that after intraperitoneal injection with α-synuclein fibrils, four of five Tg(M83 +/− : Gfap -luc +/− ) mice developed signs of neurologic dysfunction in 229 ± 17 days (black squares), whereas none of the PBS-injected control mice developed neurologic disease within 420 days (white squares). One mouse of five died 285 days after intraglossal inoculation with α-synuclein fibrils (black circles). In contrast, none of the PBS-injected mice developed neurologic disease or spontaneously died (white circles). (C) Four (black symbols) of five mice that had been inoculated intraperitoneally (IP) with α-synuclein (α-syn) fibrils continuously lost weight for 6 to 8 weeks before they developed signs of neurologic disease. The one surviving animal continuously gained weight and did not develop disease or pathology for up to 420 days after inoculation (gray symbols). (E) One (black symbols) of five mice inoculated intraglossally (IG) with α-synuclein fibrils continuously lost weight for at least 8 weeks before it died. The four mice that did not succumb to disease or die continued to gain weight (gray symbols). Mice injected intraperitoneally (D) or intraglossally (F) with PBS gained weight throughout the course of the experiment. In panels C to F, male mice are represented by squares and female mice by circles.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Staining, Sonication, Recombinant, Injection, Control

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Inoculation experiments

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques:

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Biochemical analysis of phosphorylated α-synuclein in the CNS of peripherally injected Tg(M83 +/− : Gfap -luc +/− ) mice. (A) Detection with the EP1536Y antibody, which recognizes phosphorylation at Ser129 of α-synuclein, showed that diseased mice accumulated high-molecular-weight species of Sarkosyl-insoluble aggregates of phosphorylated α-synuclein in their brains and spinal cords, whereas control mice challenged with PBS did not and showed bands only for the monomeric form of phosphorylated α-synuclein. (B) After intraglossal challenge with α-synuclein fibrils, only one mouse, animal 121, accumulated Sarkosyl-insoluble aggregates of phosphorylated α-synuclein in the brain and spinal cord, whereas all other intraglossally inoculated animals remained healthy. Molecular masses are shown in kilodaltons. Sample loading in each lane is shown by detection of actin.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Injection, Phospho-proteomics, High Molecular Weight, Control

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Immunohistochemical analysis shows neuropathology in brains of Tg(M83 +/− : Gfap -luc +/− ) mice after intraperitoneal injection with α-synuclein fibrils. (A to E and K to O) Brain sections from animals injected with α-synuclein fibrils accumulated deposits of phosphorylated α-synuclein in multiple brain regions, as detected with the pSyn#64 antibody (A to E) and the 81A antibody (K to O), which recognize phosphorylation at Ser129 of α-synuclein. (F to J and P to T) In contrast, none of the PBS-injected mice showed any detectable aggregates of phosphorylated α-synuclein in the brain. Bar = 50 μm.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Immunohistochemical staining, Injection, Phospho-proteomics

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Immunohistochemistry shows abnormal, phosphorylated α-synuclein deposits in spinal cords of Tg(M83 +/− : Gfap -luc +/− ) mice after intraperitoneal injection with α-synuclein fibrils. (A) Deposits of phosphorylated α-synuclein were present in spinal cord sections from diseased animals, as illustrated by staining with the pSyn#64 antibody. (C and E) These deposits were composed of transgenically expressed human A53T α-synuclein, as shown with the Syn211 antibody (C), and endogenously expressed mouse α-synuclein, as visualized with the D37A6 antibody (E). (B, D, and F) None of the PBS-injected animals accumulated deposits of aggregated α-synuclein in the spinal cord. Bar = 50 μm.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Immunohistochemistry, Injection, Staining

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Schematic overview showing the distribution of deposits of phosphorylated α-synuclein in the CNS of diseased Tg(M83 +/− : Gfap -luc +/− ) mice after intraperitoneal challenge with α-synuclein fibrils. (A) The distributions of abnormal deposits detected by use of two different antibodies, pSyn#64 and 81A, were comparable, and deposits could be found almost throughout the brain. However, the cerebellum was devoid of abnormal deposits of phosphorylated α-synuclein. (B) Abnormal aggregates of phosphorylated α-synuclein were also present in neurons within the gray matter of the spinal cords of diseased animals.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques:

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Phosphorylated α-synuclein can be detected in motor neurons of diseased Tg(M83 +/− : Gfap -luc +/− ) mice. (A to C) Immunofluorescence analysis showed that deposits of phosphorylated α-synuclein, as stained with the pSyn#64 antibody, were widespread in the gray matter and could also be detected in motor neurons within the ventral horn of the spinal cord in diseased Tg(M83 +/− : Gfap -luc +/− ) mice. (D to F) PBS-injected healthy control animals did not show aggregates of phosphorylated α-synuclein in their motor neurons. Motor neurons were detected with an antibody to choline O -acetyltransferase (ChAT). Nuclear staining with DAPI is shown in blue. Bar = 20 μm.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Immunofluorescence, Staining, Injection, Control

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Immunofluorescence analysis shows that deposits of phosphorylated α-synuclein colocalize with ubiquitin and p62 in the brains and spinal cords of diseased Tg(M83 +/− : Gfap -luc +/− ) mice. Phosphorylated α-synuclein, detected with the EP1536Y antibody, and ubiquitin colocalized in the brains (A to C) and spinal cords (E to G) of fibril-injected diseased mice. Similarly, phosphorylated α-synuclein, detected here with the pSyn#64 antibody, also colocalized with p62 in brains (I to K) and spinal cords (M to O) of diseased mice. (D, H, L, and P) PBS-injected healthy control animals did not show aggregation or colocalization for any of these proteins. Nuclear staining with DAPI is shown in blue. Bar = 20 μm.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Immunofluorescence, Ubiquitin Proteomics, Injection, Control, Staining

Journal: Journal of Virology

Article Title: Neuroinvasion of α-Synuclein Prionoids after Intraperitoneal and Intraglossal Inoculation

doi: 10.1128/JVI.01399-16

Figure Lengend Snippet: Gliosis in the brains of diseased Tg(M83 +/− : Gfap -luc +/− ) mice. (A) Immunofluorescence analysis of brain sections with an antibody against GFAP, a marker of astrocytes, showed that diseased animals had reactive astrogliosis in areas with deposits of phosphorylated α-synuclein, which were detected in the brain stem by use of the pSyn#64 antibody. (B) In contrast, reactive astrogliosis was not observed in brains of healthy, PBS-injected control mice. (C and D) Similarly, staining with an antibody to IBA-1, a marker of microglia, showed that diseased animals had microgliosis in areas with deposits of phosphorylated α-synuclein (C), which was not observed in brains of healthy, PBS-injected control mice (D). (E) Bioluminescence imaging revealed that Tg(M83 +/− : Gfap -luc +/− ) mice that had been challenged intraperitoneally with α-synuclein fibrils showed elevated radiance from their brains and spinal cords (left panel), caused by the increased activation of astrocytes, shortly before they developed neurologic symptoms, but this was not observed in PBS-injected control mice (right panel). After intraglossal inoculation with α-synuclein fibrils, one Tg(M83 +/− : Gfap -luc +/− ) mouse, animal 121, showed signs of reactive astrogliosis shortly before it died, at 285 days (center panel). p, photons. (F) After intraperitoneal challenge of Tg(M83 +/− : Gfap -luc +/− ) mice with α-synuclein fibrils, we measured increased levels of bioluminescence (>2 × 10 6 p/s/cm 2 /sr) from the brains of four mice (blue, green, brown, and orange circles) shortly before they developed neurologic signs of disease. One animal that was intraglossally injected with α-synuclein fibrils also showed elevated levels of bioluminescence before it died 285 days after injection (magenta circles). In contrast, healthy, PBS-injected control mice (black circles; error bars show SD [ n = 4]) and the animal that did not develop disease after intraperitoneal injection with α-synuclein fibrils (red circles) did not show increased levels of bioluminescence (<2 × 10 6 p/s/cm 2 /sr) within the period of observation. Bar = 20 μm.

Article Snippet: Human α-synuclein [Syn211] , Merck Millipore , Mouse , 121–125 , 1:100 , .

Techniques: Immunofluorescence, Marker, Injection, Control, Staining, Imaging, Activation Assay

Journal: Nature

Article Title: DAXX represents a new type of protein-folding enabler.

doi: 10.1038/s41586-021-03824-5

Figure Lengend Snippet: Fig. 1 | DAXX prevents protein misfolding and aggregation. a, b, Heat-induced luciferase inactivation (a, 5 nM) and aggregation (b, 200 nM) in presence or absence of GST, DAXX and HSP70–HSP40 at 200 nM (a) or at the indicated molar ratios (b). c, Aggregation of ATXN1(82Q) (50 nM) in the presence or absence of glutathione S-transferase (GST) or DAXX (200 nM each). Raw data for this and other gels are found in Supplementary Fig 1. PE, pelletable aggregates that were soluble with SDS; SN, supernatant. d–f, Fibrillization of α-Syn (70 μM) in the presence GST, HSP70–HSP40, HSPs (HSP70–HSP40–HSP104(A503S)) (200 nM each) and DAXX (100–400 nM) was assayed by ThT-binding (d), electron microscopy (e; red arrows, fibrils; blue arrows, large oligomers; scale bar, 100 nm), and sedimentation followed by dot blot for SDS-soluble and SDS-resistant (SR) aggregates and total α-Syn and by disuccinimidyl suberate (DSS) cross-linking for soluble oligomers (f). IB, immunoblot. g–i, Fibrillization of Aβ42 monomers (10 μM) in the absence or presence of DAXX (50–600 nM) (g, h), and viability of SH-SY5Y cells treated with Aβ42 pre-incubated with or without DAXX (i). An ATP-regeneration system was included with heat-shock proteins but not DAXX (all subsequent experiments with heat-shock proteins, but not experiments with DAXX, also contained an ATP-regeneration system). Data are mean or mean ± s.d. (n = 4 for i, and 3 for the rest) and are representative of three independent experiments. *P < 0.05, NS, not significant; unpaired Student’s t-test.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Antibodies against the following proteins/epitopes were purchased from the indicated sources: GAPDH (sc-47724,Mouse, WB, IP, IF and IHC(P)), His (sc-8036, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), GST (sc-138, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), p53DO1 (sc-126, Mouse, WB, IP, IF, IHC(P) and FCM), Mdm2 (sc-965, Mouse, WB, IP, IF and IHC(P)), DAXX (sc-8043, Mouse IP,WB, IHC(P), ELISA, IF, FCM), α-synuclein syn211 (sc-12767, Mouse, IP, WB, IHC(P), ELISA, IF, and FCM) (Santa Cruz Biotechnology); Flag (#14793, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) and DAXX (#4533S, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) (Cell Signaling Technology); p53 (PAb1620, #OP33, Mouse, WB, IP, IF; PAb240, #OP29, Mouse, WB, IP), and Mdm2 (#OP46, Mouse, WB, IP) (Calbiochem); and HA (ab137838, Rabbit, WB, ICC/IF, IP) and Luciferase (ab21176, Rabbit, ICC/IF, WB) (Abcam); βAmyloid 1-42 (#805509) (BioLegend, Mouse, WB, IHC(P), ELISA).

Techniques: Luciferase, Binding Assay, Electron Microscopy, Sedimentation, Dot Blot, Western Blot, Incubation

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Interaction of AMBRA1 with wild type (WT) α‐syn in mammalian cells. (A) Myc‐tagged AMBRA1 constructs are illustrated. FL, full length; F1–3, fragments 1–3. (B) Characterization of the site of AMBRA1 responsible for α‐syn binding. HEK293 cells were co‐transfected with Myc‐tagged AMBRA1 and RH‐tagged WT α‐syn. Protein extracts (input) were subjected to TALON purification under native conditions, in which cobalt attracts RH‐tagged protein. Purified WT α‐syn bound to the F3 portion of AMBRA1. (C–E) Proximity ligation assay (PLA) in mammalian cells. (C) No signals are recognized in the absence of the primary antibodies. (D) With the use of anti‐α‐syn (syn211) and AMBRA1 antibodies as primary antibodies for PLA, binding of endogenous α‐syn and AMBRA1 are visualized as red signals (white arrowheads). (E) Co‐transfection of WT α‐syn and AMBRA1 showed intense signals (white arrows). Bars = 20 μm.

Article Snippet: Anti‐α‐syn (syn211; 1:500) and anti‐AMBRA1 (ProSci‐Incorporated; 1:100) antibodies were utilized as primary antibodies for PLA 7 , 38 .

Techniques: Construct, Binding Assay, Transfection, Purification, Proximity Ligation Assay, Cotransfection

Journal: Movement Disorders

Article Title: Cerebrospinal Fluid Lysosomal Enzymes and Alpha-Synuclein in Parkinson's Disease

doi: 10.1002/mds.25772

Figure Lengend Snippet: GCase activity and α-syn species levels in PD patients carrying GBA1 mutations and NCs. A: Levels of GCase activity in NC PD patients and in patients carrying different GBA1 mutations. B: Total α-syn levels in CSF of NC and PD patients with GBA1 mutations. C: CSF o-α-syn levels in NC and PD patients with GBA1 mutations. D: CSF o/t-α-syn ratio levels in NC and PD patients with GBA1 mutations. GCase, β-glucocerebrosidase; α-syn, α-synuclein; PD, Parkinson's disease; NC, noncarrier; CSF, cerebrospinal fluid; o-α-syn, oligomeric α-synuclein; o/t-α-syn ratio, oligomeric/total α-synuclein ratio.

Article Snippet: Total and oligomeric CSF α-syn were measured as reported., Briefly, for CSF total α-syn, an anti-human α-syn monoclonal antibody (clone Syn211) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as primary antibody to capture a-syn species, while the anti-human α-syn polyclonal antibody FL-140 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for antigen detection.

Techniques: Activity Assay

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Immunoreactivity of phosphorylated α‐synuclein (p‐α‐syn) and AMBRA1 in the brains of patients with multiple system atrophy (MSA). Immunoreactivity for p‐α‐syn (A, C, E) and AMBRA1 (B, D, F) in glial cytoplasmic inclusions (GCIs) (A, B), neuronal cytoplasmic inclusions (NCIs) (C, D) and threads (E, F). Bars = 10 μm.

Article Snippet: Rabbit polyclonal antibodies against ULK1 (Thermo Fisher Scientific, Waltham, MA), ULK2 (Thermo Fisher Scientific), VPS34 (Thermo Fisher Scientific), beclin1 (Novus Biologicals, Littleton, CO), AMBRA1 (ProSci‐Incorporated, Poway, CA), AMBRA1 (Cell Signaling Technology, Danvers, MA), AMBRA1 (Proteintech Inc., Chicago, IL), elongin B (Abcam, Cambridge, MA), p62 (MBL, Nagoya, Japan), microtubule‐associated protein light chain 3 (LC3) (Sigma, St. Louis, MO), TNF receptor associated factor 6 (TRAF6) (Sigma) and β‐actin (Sigma), goat polyclonal antibody against Bip (Santa Cruz, Dallas, TX), rabbit monoclonal antibody against phosphorylated α‐syn (p‐α‐syn) (Abcam) and Myc (Cell Signaling Technology), and mouse monoclonal antibodies against α‐syn (Abcam, 4D6; Abcam, syn211), p‐α‐syn (Wako, Osaka, Japan; #64), dynein intermediate chain (Abcam), Flag (Sigma), RGHHHHHH His (RH) (QIAGEN, Hilden, Germany), Myc (Cell Signaling Technology) and HaloTag (Promega, Madison, WI) were utilized as primary antibodies.

Techniques:

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Double‐labeling immunofluorescence demonstrating co‐localization of AMBRA1 and p‐α‐syn in GCIs (A–C), NCIs (D–F) and threads (white arrows) (G–I). AMBRA1 appears green, and p‐α‐syn appears red. Bars = 20 μm.

Article Snippet: Rabbit polyclonal antibodies against ULK1 (Thermo Fisher Scientific, Waltham, MA), ULK2 (Thermo Fisher Scientific), VPS34 (Thermo Fisher Scientific), beclin1 (Novus Biologicals, Littleton, CO), AMBRA1 (ProSci‐Incorporated, Poway, CA), AMBRA1 (Cell Signaling Technology, Danvers, MA), AMBRA1 (Proteintech Inc., Chicago, IL), elongin B (Abcam, Cambridge, MA), p62 (MBL, Nagoya, Japan), microtubule‐associated protein light chain 3 (LC3) (Sigma, St. Louis, MO), TNF receptor associated factor 6 (TRAF6) (Sigma) and β‐actin (Sigma), goat polyclonal antibody against Bip (Santa Cruz, Dallas, TX), rabbit monoclonal antibody against phosphorylated α‐syn (p‐α‐syn) (Abcam) and Myc (Cell Signaling Technology), and mouse monoclonal antibodies against α‐syn (Abcam, 4D6; Abcam, syn211), p‐α‐syn (Wako, Osaka, Japan; #64), dynein intermediate chain (Abcam), Flag (Sigma), RGHHHHHH His (RH) (QIAGEN, Hilden, Germany), Myc (Cell Signaling Technology) and HaloTag (Promega, Madison, WI) were utilized as primary antibodies.

Techniques: Labeling, Immunofluorescence

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Interaction of AMBRA1 with wild type (WT) α‐syn in mammalian cells. (A) Myc‐tagged AMBRA1 constructs are illustrated. FL, full length; F1–3, fragments 1–3. (B) Characterization of the site of AMBRA1 responsible for α‐syn binding. HEK293 cells were co‐transfected with Myc‐tagged AMBRA1 and RH‐tagged WT α‐syn. Protein extracts (input) were subjected to TALON purification under native conditions, in which cobalt attracts RH‐tagged protein. Purified WT α‐syn bound to the F3 portion of AMBRA1. (C–E) Proximity ligation assay (PLA) in mammalian cells. (C) No signals are recognized in the absence of the primary antibodies. (D) With the use of anti‐α‐syn (syn211) and AMBRA1 antibodies as primary antibodies for PLA, binding of endogenous α‐syn and AMBRA1 are visualized as red signals (white arrowheads). (E) Co‐transfection of WT α‐syn and AMBRA1 showed intense signals (white arrows). Bars = 20 μm.

Article Snippet: Rabbit polyclonal antibodies against ULK1 (Thermo Fisher Scientific, Waltham, MA), ULK2 (Thermo Fisher Scientific), VPS34 (Thermo Fisher Scientific), beclin1 (Novus Biologicals, Littleton, CO), AMBRA1 (ProSci‐Incorporated, Poway, CA), AMBRA1 (Cell Signaling Technology, Danvers, MA), AMBRA1 (Proteintech Inc., Chicago, IL), elongin B (Abcam, Cambridge, MA), p62 (MBL, Nagoya, Japan), microtubule‐associated protein light chain 3 (LC3) (Sigma, St. Louis, MO), TNF receptor associated factor 6 (TRAF6) (Sigma) and β‐actin (Sigma), goat polyclonal antibody against Bip (Santa Cruz, Dallas, TX), rabbit monoclonal antibody against phosphorylated α‐syn (p‐α‐syn) (Abcam) and Myc (Cell Signaling Technology), and mouse monoclonal antibodies against α‐syn (Abcam, 4D6; Abcam, syn211), p‐α‐syn (Wako, Osaka, Japan; #64), dynein intermediate chain (Abcam), Flag (Sigma), RGHHHHHH His (RH) (QIAGEN, Hilden, Germany), Myc (Cell Signaling Technology) and HaloTag (Promega, Madison, WI) were utilized as primary antibodies.

Techniques: Construct, Binding Assay, Transfection, Purification, Proximity Ligation Assay, Cotransfection

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Interaction and affinity of AMBRA1 with phospho‐mimic S129E α‐syn in mammalian cells. HEK293 cells were co‐transfected with a combination of Flag‐tagged AMBRA1 and WT α‐syn, or phospho‐mimic S129E α‐syn. Inputs were subjected to immunoprecipitation (IP) using an anti‐Flag antibody. (A) Purified complexes were then analyzed by immunoblotting with an anti‐Flag antibody, showing bands of 150 kDa corresponding to the molecular weight of AMBRA1 in AMBRA1‐transfected cells (arrowhead, top). In cells transfected with AMBRA1 and WT α‐syn, an anti‐α‐syn (RH and 4D6) antibodies detected a band of 16 kDa in input and IP (arrow). A high molecular weight smear appeared in cells transfected with AMBRA1 and S129E α‐syn (asterisk, bottom). (B, C) Sensorgrams of the binding of WT α‐syn and S129E mutant to immobilized AMBRA1. Various concentrations of WT α‐syn and S129E mutant were injected onto the AMBRA1‐immobilized sensor chip. RU, resonance units.

Article Snippet: Rabbit polyclonal antibodies against ULK1 (Thermo Fisher Scientific, Waltham, MA), ULK2 (Thermo Fisher Scientific), VPS34 (Thermo Fisher Scientific), beclin1 (Novus Biologicals, Littleton, CO), AMBRA1 (ProSci‐Incorporated, Poway, CA), AMBRA1 (Cell Signaling Technology, Danvers, MA), AMBRA1 (Proteintech Inc., Chicago, IL), elongin B (Abcam, Cambridge, MA), p62 (MBL, Nagoya, Japan), microtubule‐associated protein light chain 3 (LC3) (Sigma, St. Louis, MO), TNF receptor associated factor 6 (TRAF6) (Sigma) and β‐actin (Sigma), goat polyclonal antibody against Bip (Santa Cruz, Dallas, TX), rabbit monoclonal antibody against phosphorylated α‐syn (p‐α‐syn) (Abcam) and Myc (Cell Signaling Technology), and mouse monoclonal antibodies against α‐syn (Abcam, 4D6; Abcam, syn211), p‐α‐syn (Wako, Osaka, Japan; #64), dynein intermediate chain (Abcam), Flag (Sigma), RGHHHHHH His (RH) (QIAGEN, Hilden, Germany), Myc (Cell Signaling Technology) and HaloTag (Promega, Madison, WI) were utilized as primary antibodies.

Techniques: Transfection, Immunoprecipitation, Purification, Western Blot, Molecular Weight, Binding Assay, Mutagenesis, Injection

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Protein‐protein interactions of AMBRA1 in the brains of MSA patients. Protein extracts from the cerebral cortex of normal controls and MSA patients, and cerebellar white matter of normal controls and MSA patients were immunoprecipitated using an anti‐AMBRA1 antibody. Representative cases of each brain region are shown in this figure. (A) An anti‐AMBRA1 antibody was utilized for western blot analysis of purified proteins, showing a more intense band of AMBRA1 in the cerebral cortex (lanes 2 and 6) and cerebellar white matter of MSA patients (lanes 4 and 8) than normal controls (the cerebral cortex, lanes 1 and 5; the cerebral cortex, lanes 3 and 7). A band with molecular mass of 52 kDa was observed in the cerebral cortex of MSA (arrow). (B) An anti‐α‐syn antibody (4D6) detected intense bands with a molecular weight of 16 kDa corresponding to native α‐syn in comparison with the same region between MSA patients and normal controls (white arrowhead) (right box; long time exposure). (C) For western blot analysis of immunoprecipitated proteins, a rabbit anti‐p‐α‐syn antibody was utilized. In inputs of MSA, a rabbit anti‐p‐α‐syn antibody detected a 16 kDa band. In an IP sample of the cerebral cortex of MSA, a rabbit anti‐p‐α‐syn antibody detected a band of 52 kDa, corresponding to the band detected by the anti‐AMBRA1 antibody (arrow). (D–F) PLA in the brain of MSA patients. (D) No signals were observed in the absence of primary antibodies for AMBRA1 and p‐α‐syn. Signals in GCIs (white arrows) (E) and NCIs (F). Bars = 20 μm.

Article Snippet: Rabbit polyclonal antibodies against ULK1 (Thermo Fisher Scientific, Waltham, MA), ULK2 (Thermo Fisher Scientific), VPS34 (Thermo Fisher Scientific), beclin1 (Novus Biologicals, Littleton, CO), AMBRA1 (ProSci‐Incorporated, Poway, CA), AMBRA1 (Cell Signaling Technology, Danvers, MA), AMBRA1 (Proteintech Inc., Chicago, IL), elongin B (Abcam, Cambridge, MA), p62 (MBL, Nagoya, Japan), microtubule‐associated protein light chain 3 (LC3) (Sigma, St. Louis, MO), TNF receptor associated factor 6 (TRAF6) (Sigma) and β‐actin (Sigma), goat polyclonal antibody against Bip (Santa Cruz, Dallas, TX), rabbit monoclonal antibody against phosphorylated α‐syn (p‐α‐syn) (Abcam) and Myc (Cell Signaling Technology), and mouse monoclonal antibodies against α‐syn (Abcam, 4D6; Abcam, syn211), p‐α‐syn (Wako, Osaka, Japan; #64), dynein intermediate chain (Abcam), Flag (Sigma), RGHHHHHH His (RH) (QIAGEN, Hilden, Germany), Myc (Cell Signaling Technology) and HaloTag (Promega, Madison, WI) were utilized as primary antibodies.

Techniques: Immunoprecipitation, Western Blot, Purification, Molecular Weight

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Overexpression of AMBRA1 in the degradation of S129E α‐syn in mammalian cells. (A) Increased expression of Bip, an endoplasmic reticulum stress marker, after transfection with more than 0.5 μg AMBRA1. (B) Expression levels of AMBRA1, p62, LC3, S129E α‐syn and actin in cells treated with 0.25 μg S129E α‐syn with HaloTag and 0.25 μg AMBRA1 or its empty vector. Significant decrease in the LC3‐II/LC3‐I ratio (C) and p62 in AMBRA1 overexpressing cells (D) along with a reduction of S129E α‐syn (E). The molecular weight of S129E α‐syn with HaloTag is 50 kDa. Values are expressed as means and standard deviation of independent three experiments. *P < 0.05.

Article Snippet: Rabbit polyclonal antibodies against ULK1 (Thermo Fisher Scientific, Waltham, MA), ULK2 (Thermo Fisher Scientific), VPS34 (Thermo Fisher Scientific), beclin1 (Novus Biologicals, Littleton, CO), AMBRA1 (ProSci‐Incorporated, Poway, CA), AMBRA1 (Cell Signaling Technology, Danvers, MA), AMBRA1 (Proteintech Inc., Chicago, IL), elongin B (Abcam, Cambridge, MA), p62 (MBL, Nagoya, Japan), microtubule‐associated protein light chain 3 (LC3) (Sigma, St. Louis, MO), TNF receptor associated factor 6 (TRAF6) (Sigma) and β‐actin (Sigma), goat polyclonal antibody against Bip (Santa Cruz, Dallas, TX), rabbit monoclonal antibody against phosphorylated α‐syn (p‐α‐syn) (Abcam) and Myc (Cell Signaling Technology), and mouse monoclonal antibodies against α‐syn (Abcam, 4D6; Abcam, syn211), p‐α‐syn (Wako, Osaka, Japan; #64), dynein intermediate chain (Abcam), Flag (Sigma), RGHHHHHH His (RH) (QIAGEN, Hilden, Germany), Myc (Cell Signaling Technology) and HaloTag (Promega, Madison, WI) were utilized as primary antibodies.

Techniques: Over Expression, Expressing, Marker, Transfection, Plasmid Preparation, Molecular Weight, Standard Deviation

Journal: Brain Pathology

Article Title: AMBRA1, a novel α‐synuclein‐binding protein, is implicated in the pathogenesis of multiple system atrophy

doi: 10.1111/bpa.12461

Figure Lengend Snippet: Silencing AMBRA1 function in mouse primary cultured neurons. (A) Decreased expression level of AMBRA1 by AMBRA1 siRNA (arrowhead). (B–E) Immunofluorescence analysis demonstrating transfection of AMBRA1 siRNA caused dot‐like structures of α‐syn in the cytoplasm, compared with that of control siRNA. (F) Semi‐quantitative analysis showing significantly increased number of dot‐like structures of α‐syn in the cytoplasm. LC3 appears green, and α‐syn appears red. Bars = 10 μm. **P < 0.01.

Article Snippet: Rabbit polyclonal antibodies against ULK1 (Thermo Fisher Scientific, Waltham, MA), ULK2 (Thermo Fisher Scientific), VPS34 (Thermo Fisher Scientific), beclin1 (Novus Biologicals, Littleton, CO), AMBRA1 (ProSci‐Incorporated, Poway, CA), AMBRA1 (Cell Signaling Technology, Danvers, MA), AMBRA1 (Proteintech Inc., Chicago, IL), elongin B (Abcam, Cambridge, MA), p62 (MBL, Nagoya, Japan), microtubule‐associated protein light chain 3 (LC3) (Sigma, St. Louis, MO), TNF receptor associated factor 6 (TRAF6) (Sigma) and β‐actin (Sigma), goat polyclonal antibody against Bip (Santa Cruz, Dallas, TX), rabbit monoclonal antibody against phosphorylated α‐syn (p‐α‐syn) (Abcam) and Myc (Cell Signaling Technology), and mouse monoclonal antibodies against α‐syn (Abcam, 4D6; Abcam, syn211), p‐α‐syn (Wako, Osaka, Japan; #64), dynein intermediate chain (Abcam), Flag (Sigma), RGHHHHHH His (RH) (QIAGEN, Hilden, Germany), Myc (Cell Signaling Technology) and HaloTag (Promega, Madison, WI) were utilized as primary antibodies.

Techniques: Cell Culture, Expressing, Immunofluorescence, Transfection

Journal: Nature

Article Title: DAXX represents a new type of protein-folding enabler.

doi: 10.1038/s41586-021-03824-5

Figure Lengend Snippet: Fig. 1 | DAXX prevents protein misfolding and aggregation. a, b, Heat-induced luciferase inactivation (a, 5 nM) and aggregation (b, 200 nM) in presence or absence of GST, DAXX and HSP70–HSP40 at 200 nM (a) or at the indicated molar ratios (b). c, Aggregation of ATXN1(82Q) (50 nM) in the presence or absence of glutathione S-transferase (GST) or DAXX (200 nM each). Raw data for this and other gels are found in Supplementary Fig 1. PE, pelletable aggregates that were soluble with SDS; SN, supernatant. d–f, Fibrillization of α-Syn (70 μM) in the presence GST, HSP70–HSP40, HSPs (HSP70–HSP40–HSP104(A503S)) (200 nM each) and DAXX (100–400 nM) was assayed by ThT-binding (d), electron microscopy (e; red arrows, fibrils; blue arrows, large oligomers; scale bar, 100 nm), and sedimentation followed by dot blot for SDS-soluble and SDS-resistant (SR) aggregates and total α-Syn and by disuccinimidyl suberate (DSS) cross-linking for soluble oligomers (f). IB, immunoblot. g–i, Fibrillization of Aβ42 monomers (10 μM) in the absence or presence of DAXX (50–600 nM) (g, h), and viability of SH-SY5Y cells treated with Aβ42 pre-incubated with or without DAXX (i). An ATP-regeneration system was included with heat-shock proteins but not DAXX (all subsequent experiments with heat-shock proteins, but not experiments with DAXX, also contained an ATP-regeneration system). Data are mean or mean ± s.d. (n = 4 for i, and 3 for the rest) and are representative of three independent experiments. *P < 0.05, NS, not significant; unpaired Student’s t-test.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Antibodies against the following proteins/epitopes were purchased from the indicated sources: GAPDH (sc-47724,Mouse, WB, IP, IF and IHC(P)), His (sc-8036, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), GST (sc-138, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), p53DO1 (sc-126, Mouse, WB, IP, IF, IHC(P) and FCM), Mdm2 (sc-965, Mouse, WB, IP, IF and IHC(P)), DAXX (sc-8043, Mouse IP,WB, IHC(P), ELISA, IF, FCM), α-synuclein syn211 (sc-12767, Mouse, IP, WB, IHC(P), ELISA, IF, and FCM) (Santa Cruz Biotechnology); Flag (#14793, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) and DAXX (#4533S, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) (Cell Signaling Technology); p53 (PAb1620, #OP33, Mouse, WB, IP, IF; PAb240, #OP29, Mouse, WB, IP), and Mdm2 (#OP46, Mouse, WB, IP) (Calbiochem); and HA (ab137838, Rabbit, WB, ICC/IF, IP) and Luciferase (ab21176, Rabbit, ICC/IF, WB) (Abcam); βAmyloid 1-42 (#805509) (BioLegend, Mouse, WB, IHC(P), ELISA).

Techniques: Luciferase, Binding Assay, Electron Microscopy, Sedimentation, Dot Blot, Western Blot, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Impact on DA neuron survival. (A and B) Animals received injections of AAV6–α-syn and/or sonicated PFF in SN and VTA. Quantification of TH+ cells in SN and VTA (C) and density of TH+ fibers in striatum and NAcc (D) are expressed as a percentage of the contralateral side. (E and F) Density and size of TH+ swellings. All data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the α-SYN + FIB group at the same time point. (C) Statistical analysis: SN time, F(2, 55) = 10.86, P = 0.0001; SN group, F(2, 55) = 10.32, P < 0.0002; VTA time, F(2, 58) = 4.29, P = 0.018; VTA group, F(2, 58) = 4.89, P = 0.011. (D) Time and group effect were significant in SN but not in VTA: SN time, F(2, 59) = 7.53, P = 0.0012; SN group, F(2, 59) = 14.62, P < 0.0001; VTA time, F(2, 59) = 4.46, P = 0.016; VTA group, F(2, 59) = 1.71, P = 0.189. (E) Time and group effect were significant in SN but not in VTA: SN time, F(2, 59) = 5.49, P = 0.0065; SN group, F(2, 59) = 16.18, P < 0.0001; VTA time, F(2, 59) = 2.97, P = 0.059; VTA group, F(2, 59) = 1.64, P = 0.20. (F) Significant group effect was observed in striatum, F(2, 58) = 7.25, P = 0.0016, and a significant interaction between group and time was observed in NAcc, F(4, 46) = 3.02, P = 0.0271.

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Sonication

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: α-Syn spread and impact on motor behavior. (A) Human α-syn staining shows PFF injected in SN and VTA 3 h or 3, 12, and 24 wk (w) after injection. Distribution of human α-syn derived from the AAV vector injected in SN and VTA (B) or SN (C) is shown, as indicated by the asterisks, 5–7 wk after vector injection. [Scale bars: A, B (striatum), B (SN), 1 mm.] (D and E) Changes in performance in the cylinder and stepping tests. Cylinder and stepping data both showed a significant group effect: F(2, 21) = 5.719, P = 0.0104, and F(2, 27) = 25.74, P < 0.0001, respectively.

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Staining, Injection, Derivative Assay, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Aggregation of pSyn. Representative immunostained sections show the development of intracellular pSyn+ inclusions and aggregates in SN and VTA at 3 wk (A–F), 12 wk (G–I), and 24 wk (J–L) after PFF injection for the three groups: combined AAV plus PFF (α-SYN + FIB, Left), AAV alone (α-SYN ONLY, Center), and PFF alone (FIB ONLY, Right). (Scale bars: A–C, Top, 200 μm; A–C, Bottom, 100 μm; D–F, 20 μm; G–L, Top, 200 μm; G–L, Bottom, 100 μm.). The low-power photographs were generated by stitching of images captured at 40× magnification in the microscope.

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Injection, Generated, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Development of Lewy-like synucleinopathy in nigral DA neurons over time. Triple-immunofluorescent staining shows the expression of the AAV-derived human α-syn protein (red) and the morphological appearance of the PFF-induced pSyn+ aggregates (white) in the TH+ neurons (green) in the SN. Representative photographs of individual neurons were selected from the α-SYN + FIB (Left), α-SYN ONLY (Center), and FIB ONLY (Right) groups at four different time points after PFF injection: 10 d (A–C), 3 wk (D–F), 12 wk (G–I), and 24 wk (J–L). Images were acquired as collated, 3D rendered images in the confocal microscope.

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Staining, Expressing, Derivative Assay, Injection, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Characterization of the aggregates. The pSyn-immunostained aggregates (green) observed in the SN express features characteristic of Lewy bodies (arrowheads) and Lewy neurites (arrows), staining positively for ubiquitin (red) (A–C) and thioflavin (green in E), and being resistant to proteinase K (ProtK) digestion (D and D′). Images were captured from an α-SYN + FIB–treated animal at 3 wk post-PFF injection. (Scale bars: A–C, 50 μm; D, 100 μm; D′, 50 μm; E, 10 μm.)

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Staining, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Impact on DA release assessed by in vivo amperometry. Animals received injections in the medial and lateral parts of the SN (A) of AAV–α-syn followed 4 wk later by injections of PFF (α-SYN + FIB), vehicle (α-SYN ONLY), or PFF alone (FIB ONLY) (B). (C) Chronoamperometry recordings of K+-evoked DA release were performed bilaterally from nafion-coated electrodes placed in the centrolateral part of the striatum at 10 d after PFF injection (position marked with stars on the TH-immunostained sections in D–F). (Scale bars: D–F, 1 mm for striatum and SN; D–F, Insets, 20 μm.) In the combined α-SYN + FIB group, the impact of the treatment was seen as a release curve with a lower peak and a broader kinetic profile (purple curve in G). Peak amplitude (H), reuptake rate (I), and release rate (J) were significantly reduced in the α-SYN + FIB animals (*P < 0.05, compared with control side, paired t test: peak amplitude, t(10) = 2.815, P = 0.0183; rise rate, t(9) = 2.647, P = 0.0266; and reuptake rate, t(10) = 3.097, P = 0.0113. Multivariate ANOVA showed a significant difference in peak amplitude and reuptake rate between sides, F(1, 27) = 13.50, P = 0.001, and F(1, 26) = 6.54; P = 0.017, but not in rise rate, F(1, 26) = 2.68, P = 0.114. The accumulation of p-Syn in SN and α-syn–expressing axonal processes in the striatum was much more pronounced in the combined α-SYN + FIB animals (K and N) than in the α-SYN ONLY (L and O) and the FIB ONLY (M and P) groups, accompanied by a trend toward a decrease in TH+ fiber density in the striatum in the α-SYN + FIB group (Q) F(2, 11) = 3,31, P = 0.074. (Scale bars: K–M, 200 μm; N–P, 10 μm.) The photographs in K–M were generated by stitching of images captured at 40× magnification in the microscope.

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: In Vivo, Injection, Expressing, Generated, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: TH and VMAT-2 down-regulation. (A–C) Representative photographs of triple-stained sections from the combined α-SYN + FIB group, showing marked down-regulation of TH (green) and VMAT-2 (red) in neurons containing dense p-Syn+ aggregates (white) at 10 d after PFF injection (examples marked by arrowheads). Arrows in C–C′′ mark cells where TH is down-regulated and the expression of VMAT-2 is retained. (D and E) Colabeling with the panneuronal marker Hu confirmed that part of the surviving neurons in SNc had lost their TH expression (arrowheads). In the animals treated with AAV vector alone (α-SYN ONLY), pSyn was more diffusely distributed in the cytoplasm (F) and the expression of TH and VMAT-2 was mostly retained (F′ and F′′, arrowheads). (Scale bars: A, A′, and A′′, 100 μm; B, B′, and B′′, 50 μm; C, C′, and C′′, 50 μm; D, 200 μm; E and E′, 50 μm; F, F′, and F′′, 50 μm.)

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Staining, Injection, Expressing, Marker, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Inflammatory response in SN. Total number of IBA1+ cells (A) and their staining density (B) were quantified in the SNc of animals injected with α-SYN + FIB (purple), α-SYN ONLY (red), FIB ONLY (green), or monomers (white). The differences between injected and noninjected sides, as well as between the different groups, were significant in both total number—side, F(1, 55) = 106.83, P < 0.001; group, F(3, 55) = 30.24, P < 0.001—and density—side, F(1, 55) = 73.96, P < 0.001; group, F(3, 55) = 7.56, P < 0.001, respectively. All data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the contralateral side. †P < 0.05, compared with the α-SYN + FIB group at the same time point. (C–N) Representative images of IBA1 staining for each group at different time points. (Scale bars: C–E, 500 μm; F–N, 20 μm.) (O–Q) Reactive IBA1+ microglia (red) attached to pSyn-containing cell bodies and processes (white) (arrowheads); cells with advanced pSyn pathology were frequently seen to be completely enclosed or engulfed by one or several microglial cells (arrows). The pSyn+ grains or filaments were observed also inside Iba1+ cells (double arrows). (Scale bars: 50 μm.) (R and S) Infiltration of CD4+ and CD8+ T lymphocytes in the SNc (arrowheads in R) and CD4+ expression in processes of activated microglia (arrows in R) at 3 wk after PFF injection in an animal from the α-SYN + FIB group. No similar staining was observed on the noninjected control side (Insets). (Scale bars: R, S, and Insets, 20 μm.)

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Staining, Injection, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Inflammatory response in SN pars reticulata. Total number of IBA1+ cells (A) and their staining density (B) were quantified in the SN pars reticulata of animals injected with α-SYN + FIB (purple), α-SYN ONLY (red), FIB ONLY (green), or monomers (mono; white). The differences between the injected and noninjected sides were significant in both total number, F(1, 55) = 48.61, P < 0.001, and density, F(1, 55) = 14.28, P < 0.001. All data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the contralateral side. (C–K) Representative images of IBA1 staining are presented for each group at the different time points. (Scale bars: 20 μm.)

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Staining, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: Striatal pathology. Expression of human α-syn (red) and p-Syn+ inclusions (white) was observed in axons and terminals in the striatum of animals from the α-SYN + FIB group (A) and, to a lesser extent, from the α-SYN ONLY (B) and FIB ONLY (C) groups. (Scale bars: A–C, 20 μm.) (A′′′) Some of these inclusions were resistant to proteinase K (ProtK) digestion. (D–F) The p-Syn+ grains were also observed in hypertrophic IBA1+ microglia. (Scale bars: D, 10 μm; E, 20 μm; E′ and F, 10 μm.) (G) In the PFF-injected animals p-Syn+ pathology (white) transferred to a few DARPP32+ medium spiny neurons (red). This transfer, which was detected only at 12–24 wk after PFF injection, occurred independent of the presence of DA fibers projecting to the striatum [intact side (H) and 6-OHDA–lesioned side (I)]. (Scale bars: G, H, and I, 10 μm.)

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques: Expressing, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

doi: 10.1073/pnas.1710442114

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Immunostaining was performed on 35-μm-thick frozen sections, as described elsewhere ( 9 ), using antibodies listed in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Company Host Dilution α-Syn BD Biosciences Mouse 1:1,000 α-Syn EMD Millipore Rabbit 1:1,000 α-Syn 211(human-specific) Abcam Mouse 1:1,000 pSyn 5038 (Ser129) EMD Millipore Rabbit 1:1,000 pSyn 81A (Ser129) Abcam Mouse 1:10,000 CD4 AbD Serotec Mouse 1:500 CD8 AbD Serotec Mouse 1:500 DARPP-32 Abcam Rabbit 1:1,000 Hu Abcam Mouse 1:200 IBA-1 Wako Rabbit 1:1,000 NeuN EMD Millipore Mouse 1:1,000 TH EMD Millipore Sheep 1:1,000 TH Abcam Rabbit 1:1,000 Ubiquitin Dako Rabbit 1:200 VMAT-2 Abcam Rabbit 1:5,000 Open in a separate window List of antibodies used in the study Quantification of TH + Cell Numbers, Fiber Density, and IBA1 + Microglia.

Techniques:

Journal: Scientific Reports

Article Title: Amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotides targeting α-synuclein as a novel therapy for Parkinson’s disease

doi: 10.1038/s41598-019-43772-9

Figure Lengend Snippet: ASO A19 was more efficient than ASO L19 in HEK293 cells. ( a ) qPCR analysis showing SNCA mRNA in HEK293 cells transfected with ASO A19 and ASO L19 . ASO A19 decreased the expression levels of SNCA mRNA compared to ASO L19 24 and 72 hours after transfection. Data are expressed as mean ± SEM (n = 3). *p < 0.01 by one-way ANOVA, Tukey post hoc test. ( b ) qPCR analysis of SNCA, SNCB, and SNCG mRNA in HEK293 cells transfected with ASO A19 and ASO L19 . Although ASO A19 decreased the expression levels of SNCA mRNA (see a for comparison), SNCB and SNCG levels showed no reduction. *p < 0.01 by one-way ANOVA, Tukey post hoc test. ( c , d ) Immunoblot and densitometry analysis showing that both ASO A19 and ASO L19 reduced SNCA protein levels. Proteins were extracted from HEK293 cells 72 and 96 hours after transfection of ASO A19 and ASO L19 and subjected to immunobloting. Images of blots were cropped from different parts of the same gel. Full-length gels are shown in Supplemental Fig. . Immunoblotting with anti-beta-actin was used as an internal control. Densitometry data are expressed as mean ± SEM (n = 2). ( e ) Alignment and relationship of the base sequences of four: synuclein family, human SNCA, SNCB, SNCG, and mouse Snca. The square enclosed area represents the sequence that ASO A19 can target.

Article Snippet: In brief, the following primary antibodies were used: phosphorylated α-synuclein antibody, a mouse monoclonal antibody against α-synuclein phosphorylated at Ser129 (Wako, cat # 015-25191, 1:1000 dilution) and Syn211 (Invitrogen, AHB0261, 1:1000 dilution), and a mouse monoclonal antibody that specifically recognizes human α-synuclein.

Techniques: Transfection, Expressing, Western Blot, Sequencing