α syn pffs  (StressMarq)

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: Internalized ANXA11 fibrils accumulate in lysosomes and trigger lysosomal membrane permeabilization (LMP) in neurons. a Representative confocal microscopy images of SH-SY5Y cells treated with Alexa Fluor 488-labeled ANXA11 preformed fibrils (PFFs-AF488, green) for 24 h. Cytoskeleton was stained with phalloidin (white) and nuclei with DAPI (blue). The images show intracellular localization of ANXA11 aggregates. b Flow cytometry analysis quantifying the uptake of ANXA11 PFFs by SH-SY5Y cells after 24 h treatment. c Confocal images showing the subcellular localization of internalized ANXA11 PFFs-AF647 (red) and the lysosomal marker LAMP1 (green). d Fluorescence intensity profile analysis along the white line drawn in ( c ), demonstrating the colocalization of ANXA11 PFFs with lysosomes. e Transmission electron microscopy images of SH-SY5Y cells treated with ANXA11 PFFs. Arrows indicate electron-dense aggregates within lysosomes and damaged mitochondria with disrupted cristae. f Western blot analysis of cathepsins (CSTA, CTSB, and CTSD) in cytosolic versus whole-cell lysate fractions. The presence of mature cathepsins in the cytosolic fraction upon PFFs treatment indicates lysosomal leakage. GAPDH was used as a loading control. g Representative images of Lysotracker Red staining in SH-SY5Y cells treated with or without ANXA11 PFFs, assessing lysosomal acidity and integrity. h Quantification of Lysotracker Red fluorescence intensity shown in ( g ). i Immunofluorescence images showing the recruitment of galectin-3 (GAL3, purple), a marker of lysosomal rupture, to LAMP1-positive lysosomes (green) containing ANXA11 PFFs-AF647 (red). j Fluorescence intensity profile analysis along the line in ( i ), showing the colocalization of GAL3 with ruptured lysosomes containing ANXA11 PFFs. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( h ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Membrane, Confocal Microscopy, Labeling, Staining, Flow Cytometry, Marker, Fluorescence, Transmission Assay, Electron Microscopy, Western Blot, Control, Immunofluorescence

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: ANXA11 PFFs trigger RB1CC1-dependent lysophagy and autophagic flux. a Western blot analysis of LC3 levels in SH-SY5Y cells treated with or without ANXA11 PFFs. GAPDH served as a loading control. b , c Quantification of the LC3-II/LC3-I ratio ( b ) and LC3-II/GAPDH ratio ( c ) from the blots in ( a ), indicating increased autophagic activity upon PFFs treatment. d Representative confocal images showing the recruitment of LC3 (purple) to LAMP1-positive lysosomes (green) containing ANXA11 PFFs-AF647 (red). Nuclei were stained with DAPI (blue). e Fluorescence intensity profile analysis along the white line in ( d ), confirming the colocalization of ANXA11 PFFs, LAMP1, and LC3. f Transmission electron microscopy images showing double-membrane autophagosomes engulfing electron-dense lysosomal contents (arrows), a hallmark of lysophagy, in cells treated with ANXA11 PFFs. g Western blot confirming the knockdown efficiency of RB1CC1 in SH-SY5Y cells. h Western blot analysis of LC3 turnover in Control KD and RB1CC1 KD cells treated with or without ANXA11 PFFs. i , j Quantification of LC3-II/I ( i ) and LC3-II/GAPDH ( j ) ratios from ( h ), showing that RB1CC1 knockdown prevents the PFF-induced LC3 lipidation. k Immunofluorescence staining of galectin-3 (GAL3, green) in Control KD and RB1CC1 KD cells treated with ANXA11 PFFs-AF647 (red). l Quantification of relative GAL3 fluorescence intensity in ( k ). The increase in GAL3 signal in RB1CC1 KD cells indicates an accumulation of ruptured lysosomes due to impaired clearance. m Analysis of autophagic flux using the mCherry-EGFP-LC3 reporter. Control KD cells treated with PFFs show red puncta (indicating autolysosomes, where GFP is quenched by acidity), whereas RB1CC1 KD cells retain yellow/green fluorescence. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, c ) or one-way ANOVA with Tukey’s post hoc test ( i, j, l ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Western Blot, Control, Activity Assay, Staining, Fluorescence, Transmission Assay, Electron Microscopy, Membrane, Knockdown, Immunofluorescence

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: The FTLD/ALS-linked ANXA11 D40G mutant exhibits enhanced lysosomal disruption and seeding capacity. a Representative immunofluorescence images showing the accumulation of GAL3 (purple) puncta in SH-SY5Y cells treated with WT or D40G mutant ANXA11 PFFs-AF647 (red). Lysosomes were labeled with LAMP1 (green) and nuclei with DAPI (blue). b Quantification of relative GAL3 fluorescence intensity from ( a ). The results indicate that D40G fibrils induce more severe lysosomal membrane rupture compared to WT fibrils. c Representative confocal images displaying the recruitment of LC3 (purple) to LAMP1-positive lysosomes (green) containing internalized WT or D40G ANXA11 PFFs (red). d Quantification of relative LC3 fluorescence intensity from ( c ), showing enhanced autophagic response to D40G-induced damage. e Representative images of LysoTracker Red staining in cells treated with WT or D40G ANXA11 PFFs to assess lysosomal integrity. f Quantification of LysoTracker Red fluorescence intensity from ( e ). The D40G mutant caused a more pronounced reduction in lysosomal acidity. g Representative images of HEK293T cells expressing ANXA11-GFP treated with WT or D40G ANXA11 seeds, showing induced intracellular aggregation. h Quantification of the percentage of cells containing GFP-positive inclusions from ( g ). Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, d ) or one-way ANOVA with Tukey’s post hoc test ( f, h ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Mutagenesis, Disruption, Immunofluorescence, Labeling, Fluorescence, Membrane, Staining, Expressing

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: Lysosomal damage by ANXA11 fibrils activates the p38/MK2/HSP27 signaling axis to promote lysophagy. a Western blot analysis of phosphorylated HSP27 (p-HSP27) and total HSP27 expression in SH-SY5Y cells treated with or without ANXA11 PFFs. GAPDH was used as a loading control. b , c Quantification of p-HSP27 levels normalized to total HSP27 ( b ) and GAPDH ( c ) from ( a ). d Representative immunofluorescence images showing the formation of HSP27 puncta (purple) and their colocalization with ANXA11 PFFs-AF647 (red). Phalloidin (green) stains F-actin and DAPI (blue) stains nuclei. Arrows indicate colocalization sites. e Western blot analysis of phosphorylated p38 MAPK (p-p38, T180/Y182) and total p38 MAPK levels. f , g Quantification of p-p38 levels normalized to total p38 ( f ) and GAPDH ( g ) from ( e ). h Western blot analysis of p-HSP27 and p-p38 MAPK levels in cells treated with ANXA11 PFFs in the presence or absence of the p38 MAPK inhibitor SB203580. i – l Quantification of relative protein levels from ( h ), showing that p38 inhibition effectively blocks the phosphorylation of HSP27. m Western blot analysis of phosphorylated MK2 (p-MK2, Thr334) and total MK2 levels. n , o Quantification of p-MK2 levels normalized to total MK2 ( n ) and GAPDH ( o ) from ( m ). p Western blot analysis of the p38/MK2/HSP27 pathway in cells treated with ANXA11 PFFs in the presence or absence of the MK2 inhibitor PF-3644022. q–v Quantification of phosphorylation levels from ( p ). Inhibition of MK2 significantly reduces the phosphorylation of HSP27 and upstream signaling components. w Representative immunofluorescence images of galectin-3 (GAL3, green) in cells treated with ANXA11 PFFs-AF647 (red) in the presence or absence of the p38 inhibitor PF-3644022. The increase in GAL3 puncta upon inhibition indicates exacerbated lysosomal damage due to impaired protective signaling. Data are presented as mean ± SEM. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, c, f, g, i, j, k, l, n, o, q, r, s, t, u, v ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Inhibition, Phospho-proteomics

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: Lysosomal rupture and autophagy deficiency synergistically promote the cytoplasmic seeding and neuronal propagation of ANXA11 aggregates. a Representative confocal microscopy images of SH-SY5Y cells stably expressing ANXA11-GFP. Cells were treated with ANXA11 PFFs (5 µg/mL) for 24 h, followed by exposure to the lysosomotropic agent LLOMe (1 mM) to induce lysosomal membrane permeabilization. Nuclei were counterstained with DAPI (blue). Note the marked increase in intracellular ANXA11-GFP inclusions in the combined treatment group. b Quantification of the percentage of cells containing ANXA11-GFP positive inclusions from the experiment in ( a ). c , d Dot blot analysis of cell lysates fractionated into Triton X-100 soluble and insoluble fractions. Membranes were probed with the conformation-dependent antibodies OC (targeting amyloid fibrils) ( c ) and A11 (targeting prefibrillar oligomers) ( d ). e , f Densitometric quantification of the relative signal intensity of OC ( e ) and A11 ( f ) in the insoluble fractions, normalized to controls. g Schematic illustration of the neuron-to-neuron transmission assay. Donor iPSC-derived neurons were loaded with AF647-labeled ANXA11 PFFs, washed to remove extracellular fibrils, and the conditioned medium/lysate was transferred to recipient neurons. Recipient cells were subsequently treated with LLOMe or BafA1 to assess the impact of lysosomal integrity and autophagic flux on aggregate propagation. h Representative confocal immunofluorescence images of recipient iPSC-derived neurons stained for the neuronal marker MAP2 (green). Internalized intracellular ANXA11 PFFs-AF647 are shown in red. The confocal imaging ensures that the red fluorescent signals represent genuine intracellular aggregates rather than extracellular membrane-bound particles. i Quantification of the percentage of recipient neurons containing ANXA11 PFFs-positive aggregates. The data demonstrate that both lysosomal rupture (LLOMe) and autophagy inhibition (BafA1) significantly enhance the accumulation of pathological seeds in recipient neurons. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( b, e, f, i ). Exact P -values are indicated in the graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Confocal Microscopy, Stable Transfection, Expressing, Membrane, Dot Blot, Transmission Assay, Derivative Assay, Labeling, Immunofluorescence, Staining, Marker, Imaging, Inhibition

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: ANXA11 fibril-induced lysosomal rupture triggers a cathepsin B-dependent cascade of mitochondrial dysfunction, oxidative stress, and apoptosis. a Schematic illustration of the proposed cytotoxic mechanism: lysosomal membrane permeabilization (LMP) induced by ANXA11 fibrils leads to the cytosolic release of cathepsin B, which subsequently triggers mitochondrial dysfunction, ROS accumulation, and Caspase-3-mediated apoptosis. b Representative transmission electron microscopy images of SH-SY5Y cells. Control cells exhibit healthy mitochondria with intact cristae, whereas ANXA11 PFFs-treated cells display swollen mitochondria with disrupted cristae (black arrows). c Representative confocal images showing mitochondrial morphology. Cells were stained for the mitochondrial outer membrane marker TOM20 (green), F-actin (Phalloidin, white), and nuclei (DAPI, blue). ANXA11 PFFs (red) induce mitochondrial fragmentation and network disruption. d Flow cytometric analysis of intracellular reactive oxygen species (ROS) levels using the DCFH-DA probe. Treatment with the specific Cathepsin B inhibitor CA-074Me attenuates the ROS surge induced by ANXA11 PFFs. e Representative fluorescence images of ROS generation in SH-SY5Y cells and iPSC-derived neurons. The green signal represents the fluorescence emitted by the oxidized DCF probe, visually indicating the levels of intracellular ROS. These images confirm the induction of oxidative stress upon PFFs exposure and its mitigation by lysosomal protection. f Assessment of mitochondrial membrane potential using JC-1 staining. In healthy cells, JC-1 forms red aggregates; in depolarized mitochondria, it exists as green monomers. ANXA11 PFFs induce a shift toward green fluorescence (depolarization), which is prevented by CA-074Me. g Quantification of the JC-1 aggregate-to-monomer fluorescence ratio from ( f ). h Western blot analysis of apoptotic signaling. ANXA11 PFFs increase levels of cleaved Caspase-3 (Asp175). This activation is partially blocked by inhibiting cathepsin B (CA-074Me) or autophagic flux (Bafilomycin A1). GAPDH served as a loading control. i, j Densitometric quantification of total Caspase-3 ( i ) and cleaved Caspase-3 ( j ) levels normalized to GAPDH. k Flow cytometric quantification of apoptosis using Annexin V-PE/Propidium Iodide (PI) double staining. The proportion of early (Q3) and late (Q2) apoptotic cells was significantly increased by ANXA11 PFFs but rescued by CA-074Me treatment. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( g , i , j ). Exact P -values are indicated in the graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Control, Staining, Marker, Disruption, Fluorescence, Derivative Assay, Western Blot, Activation Assay, Double Staining

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: The FTLD/ALS-linked D40G mutation exacerbates lysosomal rupture and drives a hyperactive lysophagic response in human cerebral organoids. a Schematic timeline illustrating the generation of human iPSC-derived cerebral organoids and the experimental window for ANXA11 PFFs treatment (Day 40 +). b Multiplex immunofluorescence characterization of Day 40 organoids. The tissue exhibits a complex cytoarchitecture containing PAX6 + neural progenitors (red), TUJ1 + early neurons (purple), MAP2 + mature neurons (green), TBR1 + deep-layer cortical neurons (orange), and NeuN + neuronal nuclei (white/cyan). Nuclei are counterstained with DAPI (blue). c Representative confocal images showing the internalization of AF647-labeled ANXA11 PFFs (red) into the organoid parenchyma and their specific colocalization with LAMP1 + lysosomes (green). d Assessment of lysosomal membrane permeabilization (LMP). Organoids were treated with WT or D40G ANXA11 PFFs. Note the widespread accumulation of galectin-3 (GAL3, purple) puncta on lysosomes in the D40G group, indicating severe membrane rupture. e , f Fluorescence intensity profile analyses along the white lines in ( d ), demonstrating the recruitment of GAL3 to lysosomes containing WT ( e ) and D40G ( f ) fibrils. The D40G profile shows a higher degree of signal overlap. g Evaluation of the lysophagic response. Immunostaining revealed the recruitment of the autophagy marker LC3 (purple) to LAMP1 + lysosomes (green) containing ANXA11 aggregates (red). h , i Fluorescence intensity profile analyses along the white lines in ( g ) , confirming the colocalization of LC3 with ruptured lysosomes in WT ( h ) and D40G ( i ) treated organoids. j Western blot analysis of the p38/MK2/HSP27 signaling axis in organoid lysates following treatment with WT or D40G ANXA11 PFFs. GAPDH served as a loading control. k – p Densitometric quantification of the phosphorylation levels of HSP27 ( k , l ), p38 MAPK ( m , n ), and MK2 ( o , p ). The data indicate that the D40G mutant induces a significantly more robust activation of this protective stress response pathway compared to WT fibrils. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( k , l , m , n , o , p ). Exact P -values are indicated in the graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Mutagenesis, Derivative Assay, Multiplex Assay, Immunofluorescence, Labeling, Membrane, Fluorescence, Immunostaining, Marker, Western Blot, Control, Phospho-proteomics, Activation Assay

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 expression is increased in the nigrostriatal system of a monkey model of PD induced by α-syn PFFs injection. (A) Clinical rating score of monkeys after stereotactic injection of 600 μg of α-syn PFFs or an equal volume of normal saline for 4 months. (B) Immunohistochemistry detection of TH in the STR (left) and SN (right). The TH signal intensity in the STR and the number of TH-positive neurons in the SN were lower in the PFF group. The black arrow heads indicate typical TH-positive neurons. Scale bars: 100 μm. (C) Quantitative immunohistochemistry density analysis of TH in the STR. (D) Quantitation of the ratio of TH-positive neurons in the SN in the PFF group compared with the Sham group. (E) Western blot analysis of TH, LRP1, and α-syn expression levels in the monkey STR. (F–H) Densitometric analysis of TH (F), LRP1 (G), and α-syn (H) expression levels in the STR. (I) Western blot analysis of TH, LRP1, and α-syn expression levels in the monkey SN. (J–L) Densitometric analysis of TH (J), LRP1 (K), and α-syn (L) expression levels in the SN. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Sham group (unpaired t -test). LRP1: Low-density lipoprotein receptor-related protein 1; PD: Parkinson’s disease; PFF: pre-formed fibril; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Injection, Saline, Immunohistochemistry, Quantitation Assay, Western Blot

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 expression is increased in the nigrostriatal system of a mouse model of PD induced by injection with α-Syn PFFs. (A) Visual gait analysis of walking, gait, and footprint pressure in mice after stereotactic injection with 5 μg of α-Syn PFFs or an equal volume of PBS for 4 weeks ( n = 6). (B) Normal step sequence in mice ( n = 6). (C) Average speed in mice ( n = 6). (D) Tripod support time in mice ( n = 6). (E) Immunohistochemistry staining for TH in the STR. The images in the second row are enlarged images of the areas indicated in black boxes in the upper row. The TH signal intensity in the STR was lower in the PFF group. Scale bars: 200 μm, 40 μm (enlarged). (F) Immunohistochemistry staining for TH in the SN. The images in the second row are enlarged images of the areas indicated in black boxes in the upper row, and the white arrowheads indicate typical TH-positive neurons. There were fewer TH-positive neurons in the SN in the PFF group. Scale bars: 100 μm, 20 μm (enlarged). (G) Quantitative immunohistochemistry density analysis of TH in the STR, compared with the Sham group ( n = 3). (H) Quantitation of the ratio of TH-positive neurons in the SN in the PFF group compared with the Sham group ( n = 3). (I) Western blot analysis of TH, LRP1, and α-Syn expression levels in the mouse STR after injection with 5 μg of α-Syn PFFs or an equal volume of PBS ( n = 3). (J–L) Densitometric analysis of TH (J), LRP1 (K), and α-Syn (L) expression levels in the STR. (M) Western blot analysis of TH, LRP1, and α-Syn expression levels in the mouse SN ( n = 3). (N–P) Densitometric analysis of TH (N), LRP1 (O), and α-Syn (P) expression levels in the SN ( n = 3). Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . Sham group (unpaired t -test). LRP1: Low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PD: Parkinson’s disease; PFF: pre-formed fibril; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-Syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Injection, Sequencing, Immunohistochemistry, Staining, Quantitation Assay, Western Blot

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: Exogenous α-syn PFFs upregulate LRP1 expression in PC12 cells. (A) Viability of PC12 cells incubated with different doses (0, 5, 10, 20, or 50 µg/mL) of α-Syn PFFs for 24 hours. ** P < 0.01, *** P < 0.001, vs . 0 µg/mL group. (B) Western blot analysis of LRP1 and α-syn expression levels in PC12 cells. (C, D) Densitometric analysis of LRP1 (C) and α-syn (D) expression levels. (E) Immunofluorescence analysis of LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) expression. The LRP1 and α-syn signal intensities were stronger in PFF group than in Con group. Scale bars: 40 μm. (F, G) Quantitative immunofluorescence intensity analysis of LRP1 (F) and α-syn (G). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . Con group; # P < 0.05, ## P < 0.01, vs. monomer group. Data are expressed as mean ± SD ( n = 3). Con: Control; DAPI: 4′,6-diamidino-2-phenylindole; PFF: pre-formed fibril; LRP1: low-density lipoprotein receptor-related protein 1; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Incubation, Western Blot, Immunofluorescence, Control

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 knockdown rescues the dopaminergic damage induced by exogenous α-syn PFFs. (A, B) Western blot analysis (A) and densitometric analysis (B) of TH expression levels in the STR of mice treated with PFFs. (C, D) Western blot analysis (C) and densitometric analysis (D) of TH expression levels in the SN of mice treated with PFFs. (E) Immunohistochemistry staining for TH in the STR of mice treated with PFFs and LRP1 siRNA. The decrease in relative TH intensity in STR observed in the PFF group was rescued by LRP1 siRNA treatment. The images in the second row are enlarged images of the areas indicated by black boxes in the upper row. Scale bars: 200 μm, 40 μm (enlarged). (F) Quantitative immunohistochemistry density analysis of TH in the STR. (G) Immunohistochemistry analysis of TH in the SN of mice treated with PFFs. The decreased ratio of TH-positive neurons in the SN of the Scramble + PFF group (PFF group) was rescued by LRP1 siRNA treatment. The images in the second row are enlarged images of the areas indicated by black boxes in the upper row, and the white arrowheads indicate typical TH-positive neurons. Scale bars: 100 μm, 20 μm (enlarged). (H) Quantitation of the ratio of TH-positive neurons in the SN compared with the PBS group. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Scramble + PBS group (PBS group); # P < 0.05, ## P < 0.01, vs. scramble + PFF group (PFF group). LRP1: Low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PFF: pre-formed fibril; siRNA: small interfering RNA; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Knockdown, Western Blot, Expressing, Immunohistochemistry, Staining, Quantitation Assay, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 suppression reduces the transmission of exogenous α-syn in vivo . (A) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) in the mouse STR after stereotactic injection with α-syn PFFs for 6 weeks with or without LRP1 knockdown. The increase in the relative fluorescence intensity of LRP1 and α-syn in the STR of PFFs-treated mice was rescued by LRP1 siRNA treatment. Scale bars: 100 μm. (B, C) Quantitative immunofluorescence intensity of LRP1 (B) and α-syn (C) in mouse STR. (D) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) in the SN of mice after stereotactic injection with α-syn PFFs for 6 weeks with or without LRP1 knockdown. The increase in the relative fluorescence intensity of LRP1 and α-syn in the SN of mice treated with PFFs was rescued by LRP1 siRNA treatment. The white arrowheads indicate typical cells exhibiting α-syn and LRP1 expression. Scale bars: 100 μm. (E, F) Quantitative immunofluorescence intensity of LRP1 (E) and α-syn (F) in the mouse SN. (G) Western blot analysis of LRP1 and α-syn expression levels in the mouse STR. (H, I) Densitometric analysis of LRP1 (H) and α-syn (I) expression levels in the mouse STR. (J) Western blot analysis of LRP1 and α-syn expression levels in the mouse SN. (K, L) Densitometric analysis of LRP1 (K) and α-syn (L) expression levels in the mouse SN. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, vs. Scramble + PBS group (PBS group); # P < 0.05, ## P < 0.01, vs . Scramble + PFF group (PFF group). DAPI: 4′,6-Diamidino-2-phenylindole; LRP1: low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PFF: pre-formed fibril; siRNA: small interfering RNA; SN: substantia nigra; STR: striatum; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Transmission Assay, In Vivo, Immunofluorescence, Staining, Injection, Knockdown, Fluorescence, Expressing, Western Blot, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 mediates the uptake of α-syn PFFs by PC12 cells. (A) Western blot analysis of LRP1 and α-syn expression levels in PC12 cells with or without LRP1 knockdown (10 nM siRNA) after incubation with 10 µg/mL α-syn PFFs for 24 hours. (B, C) Densitometric analysis of LRP1 (B) and α-syn (C) expression. (D) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue). The increase in the relative fluorescence intensity levels of LRP1 and α-syn in the PFF group was rescued by LRP1 siRNA treatment. Scale bars: 40 μm. (E, F) Quantitative immunofluorescence intensity analysis of LRP1 (E) and α-syn (F). Data are expressed as mean ± SD ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . Con group; # P < 0.05, ## P < 0.01, #### P < 0.0001, vs . PFF/– group. Con: Control; DAPI: 4′, 6-diamidino-2-phenylindole; PFF: pre-formed fibril; LRP1: low-density lipoprotein receptor-related protein 1; siRNA: small interfering RNA; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Western Blot, Expressing, Knockdown, Incubation, Immunofluorescence, Staining, Fluorescence, Control, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: Lysine residues in the α-syn N-terminus are vital for LRP1-mediated α-Syn internalization. (A) Heatmap of amino acids in different α-syn domains. (B) Western blot analysis of LRP1 and α-Syn levels in PC12 cells after addition of α-syn PFFs (10 µg/mL) with or without capping of lysine residues for 24 hours. (C, D) Densitometric analysis of LRP1 (C) and α-syn (D) expression levels. (E) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-Syn (red, Alexa Fluor 594), and DAPI (blue). The increase in the relative fluorescence intensity of LRP1 and α-syn in the PFF group was rescued by lysine capping of α-syn. Scale bars: 40 μm. (F, G) Quantitative immunofluorescence intensity analysis of LRP1 (F) and α-syn (G). Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, **** P < 0.0001, vs. Con group; # P < 0.05, ## P < 0.01, vs . PFF group. Con: Control; DAPI: 4′,6-diamidino-2-phenylindole; LRP1: low-density lipoprotein receptor-related protein 1; NHS: sulfo-NHS acetate; PFF: pre-formed fibril; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 shows high labeling accuracy for α-syn aggregates in vitro, compared to phosphorylated (pS129) α-syn antibody which was used as the gold standard reference staining for α-syn aggregates. (a) Structure of peptide-based probe P1 ; (b) Setup of primary hippocampal neuronal culture and induction of α-syn aggregation upon addition of PFFs; (c) Confocal microscopy of primary neuronal cells post 7 days of PFF addition. Nuclei are colored blue, total α-syn green, pS129 α-syn orange, and probe P1 in red. Scale bar represents 200 μm; (d) Probe P1 shows colocalized staining with the PS129 α-syn antibody staining. The probe labels most of the same focal locations, but with slightly larger surrounding area than the PS129 α-syn antibody hitsthis is still regarded as a positive colocalization. True positives (indicated by green arrows) are stained by both P1 and the pS129 α-syn antibody. False negatives (indicated by pink arrows) are not stained by P1 but stained by the pS129 α-syn antibody. Scale bar represents 100 μm; (e) Graph showing average positive predictive value (true positives/probe positives) of 87.4% and miss rate (false negatives/antibody positives) of 8.7%. Values on graph are given as the mean ± SEM calculated over 10 wells, with 9 fields of view each.

Article Snippet: Human recombinant α-syn monomers (SPR-321C), kinetically stable oligomers (SPR-484C), and PFFs (SPR-322C) were purchased from StressMarq Biosciences and used without further modification.

Techniques: Labeling, In Vitro, Staining, Confocal Microscopy

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 exhibits preferential binding to α-syn fibrils over α-syn monomers. (a) Fluorescent excitation and (b) fluorescent emission spectra of P1 . 2 μM probe P1 was incubated with 10 μM of various α-syn species, including monomers, A* oligomers, B* oligomers, and PFFs. Concentrations of all α-syn species are expressed at their equivalent monomer concentration. Fluorescence intensity of P1 increases significantly upon interaction with PFFs (7.6× and 16× for excitation and emission respectively); (c) Fluorescence titration curve of P1 (0.5 μM) to PFFs, showing micromolar K D ; (d) Native-PAGE images of P1 with various α-syn species, showing that P1 preferentially binds to B* oligomers and PFFs, over α-syn monomers and A* oligomers. 2 μg of P1 was incubated with 2 μg of α-syn monomers and various α-syn aggregate species and run under native PAGE conditions. In-gel fluorescence was first visualized in the 647 channel, and then the gel was subsequently imaged in the brightfield channel after staining with Coomassie blue. Red bands correspond to P1 bound to the B* oligomers and PFFs; (e) Dot-blot images of P1 (0.5 μM) with various human α-syn species, showing concentration-dependent labeling of kinetically stable oligomers and PFFs.

Article Snippet: Human recombinant α-syn monomers (SPR-321C), kinetically stable oligomers (SPR-484C), and PFFs (SPR-322C) were purchased from StressMarq Biosciences and used without further modification.

Techniques: Binding Assay, Incubation, Concentration Assay, Fluorescence, Titration, Clear Native PAGE, Staining, Dot Blot, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 shows high labeling accuracy for α-syn aggregates in vitro, compared to phosphorylated (pS129) α-syn antibody which was used as the gold standard reference staining for α-syn aggregates. (a) Structure of peptide-based probe P1 ; (b) Setup of primary hippocampal neuronal culture and induction of α-syn aggregation upon addition of PFFs; (c) Confocal microscopy of primary neuronal cells post 7 days of PFF addition. Nuclei are colored blue, total α-syn green, pS129 α-syn orange, and probe P1 in red. Scale bar represents 200 μm; (d) Probe P1 shows colocalized staining with the PS129 α-syn antibody staining. The probe labels most of the same focal locations, but with slightly larger surrounding area than the PS129 α-syn antibody hitsthis is still regarded as a positive colocalization. True positives (indicated by green arrows) are stained by both P1 and the pS129 α-syn antibody. False negatives (indicated by pink arrows) are not stained by P1 but stained by the pS129 α-syn antibody. Scale bar represents 100 μm; (e) Graph showing average positive predictive value (true positives/probe positives) of 87.4% and miss rate (false negatives/antibody positives) of 8.7%. Values on graph are given as the mean ± SEM calculated over 10 wells, with 9 fields of view each.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Labeling, In Vitro, Staining, Confocal Microscopy

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 exhibits preferential binding to α-syn fibrils over α-syn monomers. (a) Fluorescent excitation and (b) fluorescent emission spectra of P1 . 2 μM probe P1 was incubated with 10 μM of various α-syn species, including monomers, A* oligomers, B* oligomers, and PFFs. Concentrations of all α-syn species are expressed at their equivalent monomer concentration. Fluorescence intensity of P1 increases significantly upon interaction with PFFs (7.6× and 16× for excitation and emission respectively); (c) Fluorescence titration curve of P1 (0.5 μM) to PFFs, showing micromolar K D ; (d) Native-PAGE images of P1 with various α-syn species, showing that P1 preferentially binds to B* oligomers and PFFs, over α-syn monomers and A* oligomers. 2 μg of P1 was incubated with 2 μg of α-syn monomers and various α-syn aggregate species and run under native PAGE conditions. In-gel fluorescence was first visualized in the 647 channel, and then the gel was subsequently imaged in the brightfield channel after staining with Coomassie blue. Red bands correspond to P1 bound to the B* oligomers and PFFs; (e) Dot-blot images of P1 (0.5 μM) with various human α-syn species, showing concentration-dependent labeling of kinetically stable oligomers and PFFs.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Binding Assay, Incubation, Concentration Assay, Fluorescence, Titration, Clear Native PAGE, Staining, Dot Blot, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 shows specific staining of α-syn aggregates in tissues from PFF-injected mouse models. (a) Models used for ex vivo tissue labeling: PFF-injected mouse (C57BL6), where PFF was injected directly into the brain or duodenum, and transgenic mouse models overexpressing human α-syn; (b) Confocal microscopy of brain tissues after 30 days of PFF injection showing high levels of α-syn aggregation in the hippocampal and cortex regions, labeled specifically by both pS129 α-syn antibody and P1 ; (c) Confocal microscopy of duodenum tissues after 75 days of PFF injection showing low levels of α-syn aggregation in the mucosa region indicated by the white arrows. Images of tissues from control PBS-injected mice are shown in Figure S5 for comparison; (d) Confocal microscopy of brain tissue (substantia nigra) of a healthy control and of a PD patient (post-mortem), with pS129 α-syn antibody staining and P1 staining observed in the substantia nigra of the PD patient; (e) Confocal microscopy showing Lewy bodies (LB) and Lewy neurites (LN) highlighted by pS129 α-syn antibody staining in the substantia nigra of a PD patient. Higher magnification images of LB and LN show specific labeling by P1 that corresponds to the antibody staining. Nuclei in blue, α-syn in green, pS129 α-syn in orange and probe P1 in red.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Staining, Injection, Ex Vivo, Labeling, Transgenic Assay, Confocal Microscopy, Control, Comparison

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Specific α-syn antibody and probe P1 labeling of entire GI tract (esophagus, stomach, duodenum, jejunum, ileum, and colon) in transgenic mice (JAX004479). (a) Aggregated α-syn is observed in the mucosa layers and stained by both pS129 α-syn antibody and P1 (annotated with white arrows). Tissue layers are indicated by black arrows: muscularis externa (ME), submucosa (SM), and mucosa (M). Nuclei are in blue, α-syn in green, pS129 α-syn in orange, and probe in red. Scale bar represents 200 μm; (b) Total expression areas of α-syn aggregates labeled by pS129 α-syn antibody and P1 were quantified, with the highest expressions observed in the esophagus and colon; (c) High degree of colocalization (average of 0.91) was observed between pS129 α-syn antibody and probe P1 channels across all the GI tract tissues, determined by the Costes method. Manders colocalization and correlation coefficients are shown in Figure S7a ; (d) Probe positive predictive values were consistent across the respective GI tract tissues. Values on graphs are given as the mean ± SEM ( n = 3 per group); (e) P1 labeling is observed in the enteric neurons in colon tissue, marked by neuronal marker TUBB3 (green).

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Labeling, Transgenic Assay, Staining, Expressing, Marker

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Increased accumulation of α-syn aggregates across GI tissues in older transgenic mice (JAX010799). (a) Western blot showing abundance of total α-syn in brain, esophagus, duodenum, and colon of young (5-month) and old (15-month) transgenic mice (JAX010799), with higher molecular weight α-syn species observed in the older mice (indicated by red arrows); (b) Graph showing intensity of bands corresponding to α-syn, normalized to beta-actin, quantified from blots in Figure S7 . Older mice showed increased intensities of α-syn bands; Confocal images showing increased staining of aggregated α-syn in (c) esophagus and (d) colon of older transgenic mice. Tissue layers as indicated by black arrows: muscularis externa (ME), submucosa (SM), mucosa (M). Scale bar represents 200 μm; (e) Graphs showing degree of α-syn aggregation determined by pS129 α-syn antibody labeling and P1 labeling, with generally higher degrees of α-syn aggregation observed across all GI tissues in older mice. Values on graphs are given as the mean ± SEM ( n = 3 per group). Ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, determined by two-way ANOVA test.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Transgenic Assay, Western Blot, Molecular Weight, Staining, Antibody Labeling, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Longitudinal view of unrolled colon tissue of transgenic mouse showing differing levels of α-syn aggregation across tissue layers (mucosa, submucosa and muscularis externa). (a) Confocal microscopy of selected section of colon tissue from transgenic mouse, with the different tissue layers marked by white dashed lines. α-Syn aggregates on the mucosa surface are specifically labeled by pS129 α-syn antibody (orange) and P1 (red). Full view of section is shown in Figure S8 ; (b) Graph showing degree of α-syn aggregation (pS129 α-syn antibody or P1 positives, divided by α-syn positives) in the mucosa, submucosa, and muscularis externa regions. Mucosa shows lower but detectable levels of P1 staining compared to the other tissue layers; (c) Confocal microscopy of colon tissue from a transgenic mouse, flushed with P1 (15 μg/mL), followed by fixation and immunofluorescence staining with pS129 α-syn antibody (orange) and aggregated α-syn antibody, clone 5G4 (green). P1 labels α-syn aggregates in the surface mucosal layer.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Transgenic Assay, Confocal Microscopy, Labeling, Staining, Immunofluorescence