rabbit  (Proteintech)

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