Journal: Scientific Reports

Article Title: Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

doi: 10.1038/s41598-024-59090-8

Figure Lengend Snippet: Plasminogen promotes the degradation of α-syn and P-S129 α-syn in vitro, ex vivo and in vivo. ( A – D ) Recombinant α-syn protein was mixed with brain homogenates from 5 normal or 5 MPTP-treated C57BL/6 mice and incubated with vehicle or plasminogen for 6 h. ( A ) Representative WB showing α-syn levels in brain homogenates from normal mice after plasminogen incubation; ( B ) Quantitative analysis of the levels of monomeric α-syn (18 kDa) and HMW α-syn (> 33 kDa) in ( A ). ( C ) Representative WB showing α-syn levels in brain homogenates from MPTP-treated mice after plasminogen incubation; ( D ) Quantitative analysis of the levels of monomeric α-syn and HMW α-syn in ( C ). ( E, F ) Recombinant α-syn protein was incubated with saline, plasminogen, plasminogen with EACA or plasminogen with aprotinin for 6 h. ( E ) Representative WB showing α-syn levels after plasminogen incubation; ( F ) Quantitative analysis of the levels of monomeric α-syn and HMW α-syn in ( E ). For A to F , n = 2 (saline group) or 5 (other groups). ( G ) Representative WB showing α-syn expression in brain homogenates of MPTP-treated mice after plasminogen administration for 14 days; ( H ) Quantitative analysis of the relative levels of monomeric α-syn/α-tubulin and HMW α-syn/α-tubulin in ( G ). n = 7 per group in ( G , H ). ( I ) Representative WB showing α-syn expression in brain homogenates of A53T mice after plasminogen administration for 14 days; ( J ) Quantitative analysis of the relative levels of monomeric α-syn/α-tubulin and HMW α-syn/α-tubulin in ( I ). ( K ) Representative WB image of P-S129 α-syn expression in brain homogenates of A53T mice after plasminogen administration for 14 days; ( L ) Quantitative analysis of the relative levels of monomeric P-S129 α-syn /α-tubulin in ( K ). ( I – L ) n = 2 in the normal control group, n = 3 in the vehicle-treated group or plasminogen-treated group. ( M ) WB analysis with α-syn antibody (10842–1-AP) of eluates of M-280 Tosylactivated Dynabeads coupled with plasminogen or BSA. ( N – P ) Representative images of α-syn immunostaining in the substantia nigra of MPTP-treated mice after plasminogen treatment for 14 days; ( Q ) Quantitative analysis of α-syn immunostaining in ( N – P ); n = 5 in the normal control group, n = 8 in the vehicle-treated group, n = 7 in the plasminogen-treated group, scale bar = 50 μm. * P < 0.05; ** P < 0.01; *** P < 0.001. OD: optical density. MOD: mean optical density.

Article Snippet: Plasminogen was labeled with Alexa Fluor 488 dye by KMD Bioscience using the coupling method.

Techniques: In Vitro, Ex Vivo, In Vivo, Recombinant, Incubation, Saline, Expressing, Control, Immunostaining

Journal: Scientific Reports

Article Title: Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

doi: 10.1038/s41598-024-59090-8

Figure Lengend Snippet: Plasminogen promotes the degradation of hyperphosphorylated Tau ex vivo and in vivo, and of TDP-43 in vitro. ( A , B ) WB analysis showing the hyperphosphorylated Tau (p-Tau) protein levels in the brain after the administration of human plasminogen at a dose of 50 mg/kg in MPTP plus LPS-treated mice. ( A ) Representative WB analysis showing p-Tau levels in brain homogenates; ( B ) Quantitative analysis of the relative levels of monomeric (45–65 kDa) and LMW (< 45 kDa) p-Tau in ( A ), n = 5 per group. ( C – K ) NSC34 cells were exposed to OA for 24 h and subsequently treated with vehicle, plasminogen or plasminogen with EACA for 24 h. Thereafter, the cells were harvested for WB analysis of the p-Tau and total Tau (t-Tau) protein levels in the whole cells or for determination of the TDP-43 level in the cytoplasm and nucleus. ( C ) Representative WB analysis showing p-Tau levels in OA-pretreated NSC34 cells; ( D ) Quantitative analysis of the relative levels of p-Tau in ( C ). ( E ) Representative WB analysis showing t-Tau levels in OA-pretreated NSC34 cells; ( F ) Quantitative analysis of the relative levels of t-Tau in ( E ); ( G ) Quantitative analysis of the ratio of p-Tau/t-Tau in ( C , E ). ( H ) Representative WB analysis showing TDP-43 levels in the cytoplasm of OA-pretreated NSC34 cells that were left untreated (normal control) or treated with vehicle, plasminogen or plasminogen with EACA for 24 h; ( I ) Quantitative analysis of the relative TDP-43 levels shown in ( H ). ( J ) Representative WB analysis showing TDP-43 levels in the nuclei of OA-pretreated NSC34 cells treated with the control, vehicle, plasminogen or plasminogen with EACA treatments for 24 h; ( K ) Quantitative analysis of the relative TDP-43 levels shown in ( J ). n = 5 per group in ( C – K ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Plasminogen was labeled with Alexa Fluor 488 dye by KMD Bioscience using the coupling method.

Techniques: Ex Vivo, In Vivo, In Vitro, Control

Journal: Scientific Reports

Article Title: Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

doi: 10.1038/s41598-024-59090-8

Figure Lengend Snippet: Plasminogen rapidly crosses the BBB and increases plasmin activity in the blood, brain and spinal cord of MPTP-induced PD model mice after which it enters the cytoplasm and nucleus of NSC34 cells. ( A – C ) Human plasminogen levels in the blood ( A ), brain ( B ) and spinal cord ( C ) at 2, 6, 12 or 24 h after IV administration of 50 mg/kg human plasminogen. ( D ) The ratio of human plasminogen levels in the spinal cord or brain to that in the blood at 2, 6 and 12 h after IV administration of human plasminogen. ( E ) Plasmin activity in the brain 2 h after the administration of human plasminogen at a dose of 50 mg/kg in MPTP plus LPS-treated mice. n = 3 mice per group in ( A – D ); n = 5 mice per group in ( E ). ( F – I ) Human plasminogen levels in the cytoplasm ( F ) or nucleus ( G ) and plasmin activity in the cytoplasm ( H ) or nucleus ( I ) of OA-pretreated NSC34 cells after treatment with human plasminogen or human plasminogen with EACA for 24 h. ( J – M ) Quantitative comparison of the mRNA levels of the tPA gene ( J ), uPA gene ( K ), PAI-1 gene ( L ), and α2-AP gene ( M ) after human plasminogen or other treatments for 24 h in OA-pretreated NSC34 cells. n = 5 per group in ( F – M ). * P < 0.05; ** P < 0.01; *** P < 0.001. ns: no significant.

Article Snippet: Plasminogen was labeled with Alexa Fluor 488 dye by KMD Bioscience using the coupling method.

Techniques: Activity Assay, Comparison

Journal: Scientific Reports

Article Title: Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

doi: 10.1038/s41598-024-59090-8

Figure Lengend Snippet: Plasminogen enters the cytoplasm and further accumulates in the nucleus of NSC34 cells. Representative confocal micrograph of OA-pretreated NSC34 cells after administration of plasminogen-488 or plasminogen-488 plus EACA for 0, 2, 4, 6, 16, or 24 h.

Article Snippet: Plasminogen was labeled with Alexa Fluor 488 dye by KMD Bioscience using the coupling method.

Techniques:

Journal: Scientific Reports

Article Title: Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

doi: 10.1038/s41598-024-59090-8

Figure Lengend Snippet: Plasminogen decreases neurodegeneration, promotes neuroregeneration, restores normal locomotor activity in PD model mice. ( A – C ) Representative images of TH immunostaining of the substantia nigra; ( D ) Quantitative analysis of the TH immunostaining shown in ( A – C ). ( E – G ) Representative images of NF immunostaining of the striatum; ( H ) Quantitative analysis of NF immunostaining for ( E – G ). ( I – K ) Representative images of DAT immunostaining of the substantia nigra; ( L ) Quantitative analysis of the DAT immunostaining data shown in ( I – K ). Scale bar = 50 μm. ( A , E , I ): normal control group (n = 8); ( B , F , J ): vehicle-treated group (n = 10 in A – H or 12 in I – L ); ( C , G , K ) plasminogen-treated group (n = 10 in A – H or 6 in I – L ). ( M ) Representative images of the travel track in the open field test. ( N – P ) Quantitative analysis of the total distance traveled ( N ), total movement time ( O ) and number of entries into the center area ( P ). n = 8 in the normal control group, n = 10 in the vehicle-treated group, and n = 10 in the plasminogen-treated group. ( Q ) Quantitative analysis of the latency to fall in A53T mice in the rotarod test. n = 5 in the normal control group; n = 7 in the vehicle-treated group or plasminogen-treated group. * P < 0.05; ** P < 0.01; *** P < 0.001. MOD: mean optical density.

Article Snippet: Plasminogen was labeled with Alexa Fluor 488 dye by KMD Bioscience using the coupling method.

Techniques: Activity Assay, Immunostaining, Control

Journal: Scientific Reports

Article Title: Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

doi: 10.1038/s41598-024-59090-8

Figure Lengend Snippet: Schematic illustration of the possible mechanisms by which plasminogen treats PD. Administered plasminogen rapidly crosses the BBB to reach areas with α-syn or Tau aggregates/sites associated with PD pathogenesis; these areas are also where PAs, particularly tPAs, are locally expressed at high levels and further convert plasminogen to active plasmin. By binding to the lysine residues on these proteins via lysine binding sites, plasminogen/plasmin quickly decreases the aggregation of intercellular pathogenic proteins, including α-syn and Tau, fibrin, and possibly other unidentified conformationally abnormal or denatured proteins, which also help decrease further intercellular propagation of these proteins and related disease progression. In addition, through high lysine-binding affinities, plasminogen/plasmin rapidly enters the cytoplasm of neurons, possibly by endocytosis (reference and data from the current study).Moreover, plasminogen/plasmin binds to intracellular pathogenic proteins, such as α-syn, TDP-43, and Tau aggregates by recognizing the exposed lysine residues on these proteins and directly clears them via proteolytic degradation or indirectly clears them by colocalizing and possibly interacting with the intracellular UPS or ALP to further enhance their protein degradation functions. In addition, through high lysine-binding affinities, most plasminogen/plasmin gradually accumulates in the nucleus of neurons/cells, where plasminogen/plasmin degrades intranuclear pathogenic proteins such as TDP-43, as observed in the present study, and exerts gene regulatory functions, such as effects on members of the plasminogen activation system. In addition, through high lysine-binding affinities, plasminogen/plasmin also increases the abundance of mature neurotrophic molecules, such as mBDNF, by promoting the activation and maturation of pro-BDNF to exert its neuroprotective effect on damaged dopaminergic neurons, as indicated by the increased expression of DAT, TH and NF in the present study. Together, owing to its strong lysine binding properties and potent enzymatic properties, the application of plasminogen seems to simultaneously promote the ability of the PA system to promote neuroregeneration and protect against neurodegeneration, both at the molecular level, subcellular level and cellular level, eventually leading to improvements in PD pathology.

Article Snippet: Plasminogen was labeled with Alexa Fluor 488 dye by KMD Bioscience using the coupling method.

Techniques: Binding Assay, Biomarker Discovery, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Localization of the signal of dystonia-associated protein torsinA near the Golgi apparatus in cultured central neurons

doi: 10.1101/2019.12.11.872804

Figure Lengend Snippet: Hippocampal neurons were cultured from WT mice and stained for torsinA by immunocytochemistry. They were observed using a widefield microscope. A ) DIC image showing the general morphology of the cells, and fluorescence microscopy showing torsinA signal and nuclei counterstaining with Hoechst 33342 dye. Images were acquired at different focus levels. The glial cell layer is labeled 0 µm. Arrows indicate torsinA signal that is in focus, and arrowheads indicate nuclear signal that is in focus, in neurons (downward) and glial cells (upward). B ) Neurons co-stained for torsinA and the nerve terminal marker synaptophysin. They were imaged at two different levels of focus. The somatic layer was 3 µm above the dendritic, synaptophysin-positive layer. C ) Line-intensity plot obtained along the dotted line drawn in the image (inset) shown in panel B . Arrowhead indicates the staining intensity in the cytoplasm, and the broken line indicates the background intensity of the image. TorsinA and synaptophysin were imaged using secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 568 dye, respectively.

Article Snippet: Alexa Fluor 488 dye was excited using a 490-nm light-emitting diode (LED, CoolLED-Custom Interconnect, Hampshire, UK) and imaged with a filter cube (490/20-nm excitation, 510-nm dichroic long-pass, 530/40-nm emission).

Techniques: Cell Culture, Staining, Immunocytochemistry, Microscopy, Fluorescence, Labeling, Marker

Journal: bioRxiv

Article Title: Localization of the signal of dystonia-associated protein torsinA near the Golgi apparatus in cultured central neurons

doi: 10.1101/2019.12.11.872804

Figure Lengend Snippet: Neurons from WT (WT, Tor1a +/+ ), heterozygous (HET, Tor1a +/ΔE ) or homozygous (HOM, Tor1a ΔE/ΔE ) ΔE-torsinA knock-in mice were cultured, co-stained for torsinA and synaptophysin by immunocytochemistry, and observed with a widefield microscope. Neurons were obtained from the hippocampus ( A ), cerebral cortex ( B ), and striatum ( C ). DIC and synaptophysin images were acquired at the dendritic focus level, and torsinA images were acquired 2 µm higher. TorsinA and synaptophysin were visualized using secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 568 dye, respectively. There was no detectable difference in the distributions of WT-torsinA (in WT and HET mice) and ΔE-torsinA (in HET and HOM mice).

Article Snippet: Alexa Fluor 488 dye was excited using a 490-nm light-emitting diode (LED, CoolLED-Custom Interconnect, Hampshire, UK) and imaged with a filter cube (490/20-nm excitation, 510-nm dichroic long-pass, 530/40-nm emission).

Techniques: Knock-In, Cell Culture, Staining, Immunocytochemistry, Microscopy

Journal: bioRxiv

Article Title: Localization of the signal of dystonia-associated protein torsinA near the Golgi apparatus in cultured central neurons

doi: 10.1101/2019.12.11.872804

Figure Lengend Snippet: WT mouse hippocampal neurons were imaged with confocal microscopy. A ) A neuron double-stained for torsinA and the cis -Golgi marker, Golgi matrix protein of 130 kDa (GM130). B ) A neuron double-stained for torsinA and the ER marker, protein disulfide isomerase (PDI). C ) A neuron double-stained for torsinA and the nuclear envelope marker, nuclear pore complex (NPC) protein. D ) A neuron double-stained for GM130 and the ER marker, glucose-regulated protein of 78 kDa / binding immunoglobulin protein (GRP78/BiP). TorsinA and GRP78 were visualized using Alexa Fluor 488 (green channel), and GM130, PDI and NPC using Alexa Fluor 568 (red channel). In panel D , the colors in the pseudo-color images were swapped. In each grayscale image, the intensity scale was adjusted so that the minimum and maximum correspond to the 0 and 255 values on an 8-bit scale. A dotted line was drawn in the overlaid image of the two channels, and was used to generate intensity plot for each channel. The traces were normalized, with the peak intensity as 1 and the background level as 0. They were overlaid and color-coded as the antigens in the overlaid image. Asterisks indicate extensive colocalization of torsinA with GM130. Open arrows indicate torsinA peaks that partially colocalized with PDI and NPC, and GM130 peaks that partially colocalized with PDI. Arrowheads indicate diffuse torsinA staining in the cytoplasm. The dotted lines indicate the background intensity. Horizontal bar in C indicates nucleoplasmic torsinA staining, defined as staining interior to the ring of NPC signal. The main signal of endogenous WT-torsinA was in the Golgi apparatus.

Article Snippet: Alexa Fluor 488 dye was excited using a 490-nm light-emitting diode (LED, CoolLED-Custom Interconnect, Hampshire, UK) and imaged with a filter cube (490/20-nm excitation, 510-nm dichroic long-pass, 530/40-nm emission).

Techniques: Confocal Microscopy, Staining, Marker, Binding Assay