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Cited by 116Parkinson's Disease Mechanisms and TreatmentsAlzheimer's disease research and treatmentsNeuroinflammation and Neurodegeneration Mechanisms

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LRP1 is a neuronal receptor for α-synuclein uptake and spread

Open access
Chen, Kai, Martens, Yuka A., Meneses, Axel, Ryu, Daniel H., Lu, Wenyan, Raulin, Ana Caroline, Li, Fuyao, Zhao, Jing, Chen, Yixing, Jin, Yunjung, Linares, Cynthia, Goodwin, Marshall, Li, Yonghe, Liu, Chia-Chen, Kanekiyo, Takahisa, Holtzman, David M., Golde, Todd E., Bu, Guojun, Zhao, Na·2022·Molecular Neurodegeneration
DOI 10.1186/s13024-022-00560-wPMC PMC9438229
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Abstract

Background: The aggregation and spread of α-synuclein (α-Syn) protein and related neuronal toxicity are the key pathological features of Parkinson’s disease (PD) and Lewy body dementia (LBD). Studies have shown that pathological species of α-Syn and tau can spread in a prion-like manner between neurons, although these two proteins have distinct pathological roles and contribute to different neurodegenerative diseases. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP1) regulates the spread of tau proteins; however, the molecular regulatory mechanisms of α-Syn uptake and spread, and whether it is also regulated by LRP1, remain poorly understood. Methods: We established LRP1 knockout (LRP1-KO) human induced pluripotent stem cells (iPSCs) isogenic lines using a CRISPR/Cas9 strategy and generated iPSC-derived neurons (iPSNs) to test the role of LRP1 in α-Syn uptake. We treated the iPSNs with fluorescently labeled α-Syn protein and measured the internalization of α-Syn using flow cytometry. Three forms of α-Syn species were tested: monomers, oligomers, and pre-formed fibrils (PFFs). To examine whether the lysine residues of α-Syn are involved in LRP1-mediated uptake, we capped the amines of lysines on α-Syn with sulfo-NHS acetate and then measured the internalization. We also tested whether the N-terminus of α-Syn is critical for LRP1-mediated internalization. Lastly, we investigated the role of Lrp1 in regulating α-Syn spread with a neuronal Lrp1 conditional knockout (Lrp1-nKO) mouse model. We generated adeno-associated viruses (AAVs) that allowed for distinguishing the α-Syn expression versus spread and injected them into the hippocampus of six-month-old Lrp1-nKO mice and the littermate wild type (WT) controls. The spread of α-Syn was evaluated three months after the injection. Results: We found that the uptake of both monomeric and oligomeric α-Syn was significantly reduced in iPSNs with LRP1-KO compared with the WT controls. The uptake of α-Syn PFFs was also inhibited in LRP1-KO iPSNs, albeit to a much lesser extent compared to α-Syn monomers and oligomers. The blocking of lysine residues on α-Syn effectively decreased the uptake of α-Syn in iPSNs and the N-terminus of α-Syn was critical for LRP1-mediated α-Syn uptake. Finally, in the Lrp1-nKO mice, the spread of α-Syn was significantly reduced compared with the WT littermates. Conclusions: We identified LRP1 as a key regulator of α-Syn neuronal uptake, as well as an important mediator of α-Syn spread in the brain. This study provides new knowledge on the physiological and pathological role of LRP1 in α-Syn trafficking and pathology, offering insight for the treatment of synucleinopathies.

Overview

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TL;DR

Using LRP1-knockout human iPSC-derived neurons and neuronal Lrp1 conditional knockout mice, this study identifies LRP1 as a key regulator of α-synuclein neuronal uptake and spread. LRP1 primarily mediates soluble monomeric and oligomeric α-synuclein via lysine residues in the α-synuclein N-terminus, with much weaker dependence for preformed fibrils.

Key findings

  • LRP1-KO iPSNs showed significantly reduced uptake of monomeric α-synuclein (>75% reduction) and oligomeric α-synuclein (~50% reduction), while α-synuclein PFF uptake was only mildly inhibited (~5–10% reduction).
  • Capping lysine residues on α-synuclein decreased its uptake by ~50%; LRP1-KO inhibited uptake of the N-terminal α-synuclein fragment similarly to full-length α-synuclein but had no effect on the ΔN fragment, and excess non-labeled N-terminal fragment competed with full-length α-synuclein uptake whereas ΔN did not.
  • In Lrp1-nKO mice, h-α-synuclein spread to the cortex was significantly reduced compared with WT littermates, while hippocampal GFP and h-α-synuclein signals did not differ significantly between groups.
  • As validation, tau uptake was reduced by ~90% in LRP1-KO iPSNs compared with WT.

Methods

  • Human iPSCs from a healthy 83-year-old female APOE3/3 donor were edited with CRISPR/Cas9 to generate LRP1-KO isogenic lines, differentiated into iPSNs, and assayed by flow cytometry for uptake of fluorescently labeled α-synuclein monomers, oligomers, PFFs, N-terminal and ΔN fragments, lysine-capped α-synuclein, tau, and transferrin; RAP competition and electron microscopy were also used.
  • Lrp1flox/flox mice crossed with CaMKII-Cre mice on an APOE3/3 background received stereotactic hippocampal injections of AAV-synapsin-GFP-synapsin-h-α-Synuclein at 6 months of age; α-synuclein spread was assessed 3 months later by immunofluorescence and Western blotting (n = 5 WT, n = 6 Lrp1-nKO for imaging analyses).
  • Data are reported as mean ± s.d.; comparisons used unpaired t tests or one-way ANOVA with Tukey’s multiple comparison test, two-sided.

Limitations

  • The paper states that whether other cell types, such as astrocytes and microglia, can take up and spread tau and α-synuclein via LRP1 needs further investigation, and that how LRP1, HSPGs, and APOE together affect tau and α-synuclein uptake and spread requires further study.
  • Evident from the text: the iPSC experiments rely on a single parental iPSC line from one 83-year-old female APOE3/3 donor and two LRP1-KO clones, and the in vivo spread assay uses one injection site, one age, and one post-injection time point in an APOE3/3 background.
  • Evident from the text: because LRP1-KO only mildly reduced PFF uptake, the strongest conclusions concern soluble monomeric and oligomeric α-synuclein rather than fibrillar α-synuclein.

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Registry

Crossref · 10.1186/s13024-022-00560-w

Cites

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Figures

Fig. 1 Generation and validation of human induced pluripotent stem cells (iPSCs)-derived neurons (iPSNs) with LRP1 gene knockout (LRP1-KO). a Schematic diagram of the workflow for LRP1-KO iPSC generation, neural differentiation, and protein uptake assays. LRP1-KO iPSC colonies were obtained using CRISPR/Cas9 gene editing stratagy. Neural progenitor cells (NPCs) were then induced from the iPSCs and further differentiated into iPSNs. On day 14 to 16 of iPSN differentiation, neurons were treated with fluorescently labeled proteins and the uptake was measured by flow cytometry. b Immunofluorescence images showing NPCs from all three lines (WT, LRP1-KO#1, and LRP1-KO#2) were positive for neural precursor marker, Nestin. Scale bars, 100 μm. c, Immunofluorescence images of iPSNs from all three cell lines were positive for neuronal marker, Tuj1. Scale bars, 100 μm. d and e, Detection and quantification of LRP1 protein levels in WT and LRP1-KO iPSNs via Western blotting. f and g, Endocytosis of human tau in WT and LRP1-KO iPSNs measured by flow cytometry (100 nM, 3 h of treatment). Experiments in (f and g) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; ***P < 0.001
Fig. 1 Generation and validation of human induced pluripotent stem cells (iPSCs)-derived neurons (iPSNs) with LRP1 gene knockout (LRP1-KO). a Schematic diagram of the workflow for LRP1-KO iPSC generation, neural differentiation, and protein uptake assays. LRP1-KO iPSC colonies were obtained using CRISPR/Cas9 gene editing stratagy. Neural progenitor cells (NPCs) were then induced from the iPSCs and further differentiated into iPSNs. On day 14 to 16 of iPSN differentiation, neurons were treated with fluorescently labeled proteins and the uptake was measured by flow cytometry. b Immunofluorescence images showing NPCs from all three lines (WT, LRP1-KO#1, and LRP1-KO#2) were positive for neural precursor marker, Nestin. Scale bars, 100 μm. c, Immunofluorescence images of iPSNs from all three cell lines were positive for neuronal marker, Tuj1. Scale bars, 100 μm. d and e, Detection and quantification of LRP1 protein levels in WT and LRP1-KO iPSNs via Western blotting. f and g, Endocytosis of human tau in WT and LRP1-KO iPSNs measured by flow cytometry (100 nM, 3 h of treatment). Experiments in (f and g) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; ***P < 0.001CC BY · PubMed Central
Fig. 2 LRP1 regulates α-Syn uptake in iPSNs. a and b, α-Syn uptake in WT and LRP1-KO iPSNs measured by flow cytometry (100 nM, 3 h of treatment). c Representative images of WT or LRP1-KO iPSNs after α-Syn uptake. Scale bars, 20 μm. d and e, Transferrin (Tfn) uptake in WT and LRP1-KO iPSNs measured by flow cytometry (300 nM, 3 h of treatment). f Uptake of α-Syn and Tfn in the presence of increasing concentrations of RAP. g EM images showing the structure of α-Syn oligomers and preformed fibrils (PFFs) used in panel h. Scale bars, 200 nm. h Uptake of α-Syn oligomers and PFFs in WT and LRP1-KO iPSNs (100 nM monomer equivalent, 3 h of treatment). All experiments in (a, b, d, e, f and h) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2 LRP1 regulates α-Syn uptake in iPSNs. a and b, α-Syn uptake in WT and LRP1-KO iPSNs measured by flow cytometry (100 nM, 3 h of treatment). c Representative images of WT or LRP1-KO iPSNs after α-Syn uptake. Scale bars, 20 μm. d and e, Transferrin (Tfn) uptake in WT and LRP1-KO iPSNs measured by flow cytometry (300 nM, 3 h of treatment). f Uptake of α-Syn and Tfn in the presence of increasing concentrations of RAP. g EM images showing the structure of α-Syn oligomers and preformed fibrils (PFFs) used in panel h. Scale bars, 200 nm. h Uptake of α-Syn oligomers and PFFs in WT and LRP1-KO iPSNs (100 nM monomer equivalent, 3 h of treatment). All experiments in (a, b, d, e, f and h) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001CC BY · PubMed Central
Fig. 3 LRP1 regulates α-Syn uptake via lysine residues in the N-terminus of α-Syn. a Schematic diagram of α-Syn domains highlighting the lysine residues (K). b Uptake of α-Syn and lysine-capped α-Syn in WT iPSNs. c, Uptake of α-Syn-488, N-α-Syn-488 and ΔN-α-Syn-488 in WT and LRP1-KO iPSNs. d Uptake of α-Syn-488 in the presence of excessive non-labeled α-Syn N-terminus (N-α-Syn) or α-Syn lacking N-terminus (ΔN-α-Syn) fragments. All experiments in (b–d) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data in (b) was analyzed by unpaired two-sided t-test. ***P < 0.001. Data in (c and d) were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3 LRP1 regulates α-Syn uptake via lysine residues in the N-terminus of α-Syn. a Schematic diagram of α-Syn domains highlighting the lysine residues (K). b Uptake of α-Syn and lysine-capped α-Syn in WT iPSNs. c, Uptake of α-Syn-488, N-α-Syn-488 and ΔN-α-Syn-488 in WT and LRP1-KO iPSNs. d Uptake of α-Syn-488 in the presence of excessive non-labeled α-Syn N-terminus (N-α-Syn) or α-Syn lacking N-terminus (ΔN-α-Syn) fragments. All experiments in (b–d) were performed in technical duplicates or triplicates over three independent experiments. All data are expressed as mean ± s.d. with individual data points shown. Data in (b) was analyzed by unpaired two-sided t-test. ***P < 0.001. Data in (c and d) were analyzed by One-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001CC BY · PubMed Central
Fig. 4 Neuronal Lrp1 knockout reduces α-Syn spread in vivo. a Schematic drawing for the stereotactic injection of AAV-synapsin-GFP-synapsin-h-α-Synuclein into the neuronal Lrp1 knockout (Lrp1-nKO) mice and wild type (WT) littermate controls, and the experimental workflow. b and c, Western blotting showing the endogenous Lrp1 protein in the cortex of WT (n = 6) and Lrp1-nKO mice (n = 6). d Representative sections showing GFP and h-α-Syn signals in mouse brains. Dotted line marks the outline of each section. Scale bars, 500 μm. e Representative images showing GFP and h-α-Syn signals in the hippocampus region from WT and Lrp1-nKO mice. Scale bars, 50 μm. f Quantitative analysis of GFP intensity in hippocampus from WT or Lrp1-nKO mice. g Quantitative analysis of h-α-Syn immunofluorescence intensity in hippocampus from WT or Lrp1-nKO mice. h Representative images showing h-α-Syn spreading to the cortex region from WT or Lrp1-nKO mice. Scale bars, 50 μm. i Quantitative analysis of h-α-Syn immunofluorescence intensity in the cortex from WT or Lrp1-nKO mice. Experiments in (e–i) n = 5 mice (3 males and 2 females) for WT and n = 6 mice (3 males and 3 females) for Lrp1-nKO mice. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by unpaired two-sided t-test. NS, not significant; *P < 0.05, ***P < 0.001
Fig. 4 Neuronal Lrp1 knockout reduces α-Syn spread in vivo. a Schematic drawing for the stereotactic injection of AAV-synapsin-GFP-synapsin-h-α-Synuclein into the neuronal Lrp1 knockout (Lrp1-nKO) mice and wild type (WT) littermate controls, and the experimental workflow. b and c, Western blotting showing the endogenous Lrp1 protein in the cortex of WT (n = 6) and Lrp1-nKO mice (n = 6). d Representative sections showing GFP and h-α-Syn signals in mouse brains. Dotted line marks the outline of each section. Scale bars, 500 μm. e Representative images showing GFP and h-α-Syn signals in the hippocampus region from WT and Lrp1-nKO mice. Scale bars, 50 μm. f Quantitative analysis of GFP intensity in hippocampus from WT or Lrp1-nKO mice. g Quantitative analysis of h-α-Syn immunofluorescence intensity in hippocampus from WT or Lrp1-nKO mice. h Representative images showing h-α-Syn spreading to the cortex region from WT or Lrp1-nKO mice. Scale bars, 50 μm. i Quantitative analysis of h-α-Syn immunofluorescence intensity in the cortex from WT or Lrp1-nKO mice. Experiments in (e–i) n = 5 mice (3 males and 2 females) for WT and n = 6 mice (3 males and 3 females) for Lrp1-nKO mice. All data are expressed as mean ± s.d. with individual data points shown. Data were analyzed by unpaired two-sided t-test. NS, not significant; *P < 0.05, ***P < 0.001CC BY · PubMed Central

Verification

Claims the paper makes

18 Own result · 16 Own interpretation · 9 Own method · 6 Not checked yet · 4 Supported · 3 No full text of the sources · 2 Partly supported · 1 No basis given · accuracy not yet measured

Verify all 8 references — free account
  • Probably not a substantial claim. 9
  • It may state two separate claims as one. 13
19 of 19

  • The paper relies on this without support.

    α-Syn has a similar high content (10%) of lysines as tau.

    “Notably, α-Syn has a similar high content (10%) of lysines as tau.”

  • The paper relies on this without support.

    The underlying cellular mechanisms of α-Syn uptake and spread remain poorly understood.

    “However, the underlying cellular mechanisms of α-Syn uptake and spread remains poorly understood.”

  • The paper relies on this without support.

    Secreted soluble α-Syn can be taken up by different cells.

    “Importantly, these secreted soluble α-Syn can be taken up by different cells.”

  • The paper relies on this without support.

    Capping lysine residues on α-Syn with sulfo-NHS acetate specifically blocks lysine residues without otherwise altering α-Syn uptake.

    “we capped the amines of lysines on α-Syn with sulfo-NHS acetate and then measured the internalization of α-Syn in the iPSNs.”

  • The paper relies on this without support.

    The N-α-Syn and ΔN-α-Syn fragments are properly folded and their uptake reflects the role of the N-terminus.

    “We employed the α-Syn protein fragments containing only the N-terminus (N-α-Syn) or lacking the N-terminus (ΔN-α-Syn) of full-length α-Syn (Fig. 3a).”

  • The paper relies on this without support.

    LRP1-nKO mice have no compensatory mechanisms that restore LRP1 function or alter α-Syn spread independently of LRP1 deletion.

    “we used the conditional neuronal Lrp1 knockout mice [38] by crossing Lrp1flox/flox mice with CaMKII-Cre mice to generate Lrp1-nKO (Lrp1flox/flox; Cre+/−) and WT littermate controls (Lrp1flox/flox; Cre−/−).”
6 of 6

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Percentages, sums and reported differences re-computed from the paper’s own numbers.

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1 of 1

Citations

Cites

Show all 64 references

Research works printed but not linked: 2

  1. Kanekiyo T, Bu G. The low-density lipoprotein receptor-related protein 1 and amyloid-beta clearance in Alzheimer’s disease. Front Aging Neurosci. 2014;6:93. · DOI · Paper page
  2. Jin Y, Li F, Sonoustoun B, Kondru NC, Martens YA, Qiao W, Heckman MG, Ikezu TC, Li Z, Burgess JD, et al. APOE4 exacerbates alpha-synuclein seeding activity and contributes to neurotoxicity in Alzheimer’s disease with Lewy body pathology. Acta Neuropathol. 2022;143:641. · DOI · Paper page

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Metadata

Authors
Chen, K., Martens, Y. A., Meneses, A., Ryu, D. H., Lu, W., Raulin, A. C., Li, F., Zhao, J., Chen, Y., Jin, Y., Linares, C., Goodwin, M., Li, Y., Liu, C.-C., Kanekiyo, T., Holtzman, D. M., Golde, T. E., Bu, G., & Zhao, N.
Affiliations
  • Mayo Clinic (US)
  • University of Florida (US)
  • Washington University in St. Louis (US)
Corresponding author
  • Bu, Guojun
  • Zhao, Na
ORCID
Venue
Molecular Neurodegeneration
Publisher
Springer Science and Business Media LLC
Published
2022-09-02
Volume
17
Issue
1
Pages
57-57
ISSN
1750-1326 (ISSN-L)
DOI
10.1186/s13024-022-00560-w
PMC
PMC9438229
OpenAlex
W4294168387
Cited by
115
References
64
Citations by year
2026: 31 · 2025: 40 · 2024: 26 · 2023: 15 · 2022: 3
FWCI
11.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Neurodegeneration (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Rare & Special e-Zone (The Hong Kong University of Science and Technology) (submittedVersion)
Funders
  • NIH (P01NS074969, U19AG069701)
  • Mayo Alzheimer’s Disease Research Center Developmental Grant
  • Lewy Body Dementia Center Without Walls (U54NS110435)
Topics
  • Health Sciences › Medicine › Clinical Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
alpha-synuclein, Parkinson's disease, dementia with Lewy bodies, protein uptake, prion-like spread, lysine residues, N-terminus, preformed fibrils, oligomers, lysosomal degradation, iPSC-derived neurons, neurodegeneration, tau spread, isogenic iPSC lines
MeSH terms
Animals, Humans, Infant, Parkinson Disease, Synapsins, tau Proteins, Low Density Lipoprotein Receptor-Related Protein-1, Mice, alpha-Synuclein, Induced Pluripotent Stem Cells

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