Endothelial LRP1 protects against neurodegeneration by blocking cyclophilin A - PubMed (original) (raw)

. 2021 Apr 5;218(4):e20202207.

doi: 10.1084/jem.20202207.

Yaoming Wang # 1, Qingyi Ma # 1, Abhay P Sagare 1, Axel Montagne 1, Mikko T Huuskonen 1, Sanket V Rege 1, Kassandra Kisler 1, Zhonghua Dai 1, Jakob Körbelin 2, Joachim Herz 3 4, Zhen Zhao # 1, Berislav V Zlokovic # 1

Affiliations

Endothelial LRP1 protects against neurodegeneration by blocking cyclophilin A

Angeliki Maria Nikolakopoulou et al. J Exp Med. 2021.

Abstract

The low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic and cell signaling transmembrane protein. Endothelial LRP1 clears proteinaceous toxins at the blood-brain barrier (BBB), regulates angiogenesis, and is increasingly reduced in Alzheimer's disease associated with BBB breakdown and neurodegeneration. Whether loss of endothelial LRP1 plays a direct causative role in BBB breakdown and neurodegenerative changes remains elusive. Here, we show that LRP1 inactivation from the mouse endothelium results in progressive BBB breakdown, followed by neuron loss and cognitive deficits, which is reversible by endothelial-specific LRP1 gene therapy. LRP1 endothelial knockout led to a self-autonomous activation of the cyclophilin A-matrix metalloproteinase-9 pathway in the endothelium, causing loss of tight junctions underlying structural BBB impairment. Cyclophilin A inhibition in mice with endothelial-specific LRP1 knockout restored BBB integrity and reversed and prevented neuronal loss and behavioral deficits. Thus, endothelial LRP1 protects against neurodegeneration by inhibiting cyclophilin A, which has implications for the pathophysiology and treatment of neurodegeneration linked to vascular dysfunction.

© 2021 Nikolakopoulou et al.

PubMed Disclaimer

Conflict of interest statement

Disclosures: J. Körbelin reported personal fees from Boehringer Ingelheim Pharma outside the submitted work; in addition, J. Körbelin had a patent number 10696717 issued and a patent to 10688151 issued. J. Herz reported a patent to 7192714 issued and a patent to 7056688 issued. No other disclosures were reported.

Figures

Figure 1.

Figure 1.

Endothelial LRP1 expression and deletion in mice. (A–C) LRP1 immunoblotting (#92544 mAb; Abcam; A), Lrp1 mRNA quantitative real-time PCR (B), and relative Lrp1 mRNA gene expression normalized to Gapdh (housekeeping gene; C) in brain endothelial cells cultured from Lrp1lox/lox littermate control mice (control), Lrp1lox/+; Tie2-Cre (lox/+) and Lrp1lox/lox; Tie2-Cre (lox/lox) mice. In C, for relative gene expression calculations, we used the 2^(−ΔCt) method, where Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level). With respect to the ΔCt of the 2^(−ΔCt) method, ΔCt is the difference in the threshold cycles between Lrp1 and Gapdh genes. (D–F) LRP1 immunoblotting (D), Lrp1 mRNA quantitative real-time PCR (E), and relative Lrp1 mRNA gene expression normalized to Gapdh (F) in pericytes cultured from Lrp1lox/lox littermate control mice (control), Lrp1lox/+; Tie2-Cre (lox/+), and Lrp1lox/lox; Tie2-Cre (lox/lox) mice. Calculations in F were the same as in C. (G) Representative confocal images of triple immunofluorescent staining for LRP1, CD31 (endothelium), and CD13 (pericytes) showing expression of LRP1 in endothelium in isolated cortical capillaries from Lrp1lox/lox control mice and its deletion from endothelium in capillaries from Lrp1lox/lox; Tie2-Cre (lox/lox) mice; purple, merged LRP1 and CD31 (endothelium); yellow, merged LRP1 and CD13 (pericytes). No changes in expression of LRP1 in pericytes were observed between control and Lrp1lox/lox; Tie2-Cre (lox/lox) mice. Scale bar = 10 µm. (H) High-magnification orthogonal views showing colocalization of LRP1 with endothelial marker CD31 in brain capillaries from Lrp1lox/lox control mice (left) and loss of LRP1 from endothelium in brain capillaries from Lrp1lox/lox; Tie2-Cre mice (right). CD13, pericyte marker. Scale bar = 15 µm. (I) Representative confocal images of triple immunofluorescent staining for LRP1, GLUT1 (endothelium), and CD13 (pericytes) showing expression of LRP1 in endothelium in isolated cortical capillaries from Lrp1lox/lox control mice and its deletion from endothelium in capillaries from Lrp1lox/lox; Tie2-Cre (lox/lox) mice; purple, merged LRP1 and GLUT1 (endothelium); yellow, merged LRP1 and CD13 (pericytes). No changes in expression of LRP1 in pericytes were observed between control and Lrp1lox/lox; Tie2-Cre (lox/lox) mice. Scale bar = 10 µm. In C and F, significance was determined by one-way ANOVA followed by Bonferroni post hoc test; ns, not significant; ****, P < 0.0001.

Figure 2.

Figure 2.

BBB breakdown, neuron loss, and behavioral deficits after endothelial-specific Lrp1 deletion. (A) Immunostaining for IgG (left, purple), fibrinogen (right, purple), and lectin+ endothelium (green) shows perivascular capillary leakages of blood-derived proteins in the cortex of 2-mo-old Lrp1lox/lox; Tie2-Cre mice compared with Lrp1lox/lox littermate controls. Scale bar = 20 µm. (B and C) Quantification of IgG (B) and fibrin (C) deposits in the cortex (Ctx) and hippocampus (Hp) in 2- and 4-mo-old Lrp1lox/lox; Tie2-Cre mice and Lrp1lox/lox controls. Mean ± SEM, n = 5 mice/group. (D and E) Capillary leakages of intravenously administered 40 kD FITC-dextran in the cortex of 2-mo-old Lrp1lox/lox; Tie2-Cre mice compared with Lrp1lox/lox control mice (D), and quantification of the BBB permeability–surface area product (PS) to 40 kD FITC-dextran in 2- and 4-mo-old mice (E). Scale bar = 50 µm. Mean ± SEM, n = 6 mice/group. (F and G) Cortical uptake of intravenously administered Alexa Fluor 555–cadaverine (red) in 2-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice (F), and quantification of cadaverine cortical uptake in 2- and 4-mo-old mice (G). Scale bar = 25 µm. White, lectin+ endothelium. Mean ± SEM, n = 5 mice/group. (H–J) NeuN+ neurons and SMI312+ neurites (H), and quantification of NeuN+ neurons and SMI312+ neurites in the cortex and hippocampus in 2- and 4-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice (I and J). Scale bar = 50 µm. Mean ± SEM, n = 5 mice/group. (K–N) Burrowing (K), nest construction (L), novel object location (M), and recognition (N) in 2- and 4-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice. Mean ± SEM, n = 12 (K and L) and n = 15–17 (M and N) mice/group. In B, C, E, G, and I–N, significance was determined by one-way ANOVA followed by Bonferroni post hoc test; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. In B, C, E, and G, asterisks above red dots indicate comparisons with control group.

Figure S1.

Figure S1.

Intact BBB and microvascular pattern in Lrp1lox/lox; Tie2-Cre mice at P8, intact microvascular density in 2-mo-old Lrp1lox/lox; Tie2-Cre mice, and BBB breakdown in 1-mo-old Lrp1lox/lox; Tie2-Cre mice. (A) Confocal images of lectin+ endothelial microvascular profiles in the cortex (green) and retina (red) in 8-d-old (P8) Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox littermate control pups injected intravenously with Alexa Fluor 555–conjugated cadaverine show no BBB leakage of tracer into the cortex (red) or retina (green). Representative images shown are from three mice per group. Scale bars = 200 µm. (B and C) Microvascular length (B) and capillary branching points (C) determined in 10-µm-thick cortical tissue sections from P8 Lrp1lox/lox and Lrp1lox/lox; Tie2-Cre mice and expressed per square millimeter of tissue. (D) Microvascular length in the cortex of 2-mo-old Lrp1lox/lox and Lrp1lox/lox; Tie2-Cre mice and expressed per square millimeter of tissue. (E and F) Quantification of fibrinogen deposits (E) and leakage of Alexa Fluor 555–conjugated cadaverine tracer (F) deposits in the cortex of 1-mo-old Lrp1lox/lox; Tie2-Cre mice compared with Lrp1lox/lox controls. In B–F, data represent mean ± SEM, n = 3–5 mice/group; ns, nonsignificant by Student’s t test; **, P < 0.01; ***, P < 0.001.

Figure S2.

Figure S2.

Additional characterization of Lrp1lox/lox; Tie2-Cre mice. (A and B) Representative confocal microscopy images of CD13+ pericytes (magenta) and lectin+ endothelium (blue) in the cortex of 4-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox littermate control mice (A), and quantification of pericyte coverage of brain capillary (<6 µm in diameter) lectin+ endothelial profiles (B). Scale bar = 25 µm. Mean ± SEM, n = 5 mice/group. (C and D) Representative confocal images of aquaporin 4+ astrocyte endfeet (green) and lectin+ endothelium (purple) in the cortex of 4-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox littermate control mice (C) and quantification of astrocytic endfeet aquaporin 4+ coverage of brain capillary (<6 µm in diameter) lectin+ endothelial profiles (D). Scale bar = 25 µm. Mean ± SEM, n = 5 mice/group. (E and F) Representative confocal images of α-syntrophin+ astrocytic endfeet (green) and lectin+ endothelium (purple) in the cortex of 4-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox littermate control mice (E) and quantification of astrocytic endfeet α-syntrophin+ coverage of brain capillary (<6 µm in diameter) lectin+ endothelial profiles (F). Scale bar = 25 µm. Mean ± SEM, n = 5 mice per group. (G and H) Immunoblotting (G) and relative abundance of endothelial major facilitator superfamily domain–containing protein 2 (MSFD2a) using β-actin as a loading control (H) in isolated brain microvessels from 4-mo-old Lrp1lox/lox; Tie2-Cre mice and Lrp1lox/lox littermate controls. (I and J) Immunoblotting (I) and relative abundance of endothelial glucose transporter 1 (GLUT1) using β-actin as a loading control (J) in isolated brain microvessels from 4-mo-old Lrp1lox/lox; Tie2-Cre mice and Lrp1lox/lox littermate controls. In H and J, mean ± SEM, n = 3 mice per group. In B, D, F, H, and J, ns, nonsignificant by Student’s t test.

Figure S3.

Figure S3.

Neuronal degeneration in 3-mo-old Lrp1lox/lox; Tie2-Cre mice after endothelial-specific Lrp1 deletion. (A and B) Quantification of NeuN+ neurons (A) and SMI312+ neurites (B) in the cortex (Ctx) and hippocampus (Hp) of 3-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice. Mean ± SEM, n = 5 mice/group. Significance by Student’s t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Figure 3.

Rescue of vascular and neuronal phenotype in Lrp1lox/lox; Tie2-Cre mice by endothelial-specific LRP1 gene replacement. (A and B) Endothelial expression of GFP (left, green), HA-tagged mLRP1 (right, green), and lectin+ endothelium (purple) in the cortex of 4-mo-old Lrp1lox/lox; Tie2-Cre mice after AAV2-BR1_-GFP_ (left) or AAV2-BR1_-m_LRP1 (LRP1 minigene, right) treatment (A), and quantification of HA-mLRP1+ area colocalized with lectin+ endothelial capillary profiles (<6 µm in diameter) in the cortex (Ctx) and hippocampus (Hp; B). Scale bar = 25 µm. Mean ± SEM; n = 3 mice/group. (C–E) IgG (left, purple) and fibrinogen (right, purple) perivascular capillary deposits (C) and quantification (D and E) in 4-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice after AAV2-BR1_-GFP_ (top) or AAV2-BR1_-m_LRP1 (bottom) treatment. Green, lectin+ endothelium. Scale bar = 20 µm. Mean ± SEM, n = 14 mice/group. (F) High-magnification confocal images from Lrp1lox/lox; Tie2-Cre mouse treated with AAV2-BR1_-m_LRP showing that vessel segments that reexpress HA-mLRP1 (asterisks) were no longer leaky compared with a vessel segment that does not express HA-mLRP1 (arrow) that leaks fibrinogen. Scale bar = 10 µm. (G–I) NeuN+ neurons and SMI312+ neurites (G) and quantification of NeuN+ neurons and SMI312+ neurites in the cortex and hippocampus of 4-mo-old Lrp1lox/lox and Lrp1lox/lox; Tie2-Cre mice after AAV2-BR1_-GFP_ (top panels) or AAV2-BR1_-m_LRP1 (bottom panels) treatment (H and I). Scale bar = 50 µm. Mean ± SEM, n = 14 mice/group. (J–M) Burrowing (J), nest construction (K), novel object location (L), and recognition (M) in 4-mo-old Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice after AAV2-BR1_-GFP_ or AAV2-BR1_-m_LRP1 treatment. Mean ± SEM; n = 14 mice/group. In B, significance was determined by Student’s t test. In D, E, and H–M, significance was determined by one-way ANOVA followed by Bonferroni post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 4.

Figure 4.

Activation of CypA-MMP9 pathway after Lrp1 endothelial loss. (A–D) ZO-1 (A, purple), occludin (B, purple), and claudin 5 (C, purple) colocalization with endothelial lectin (green) in the cortex of 2-mo-old Lrp1lox/lox; Tie2-Cre mice and Lrp1lox/lox controls, and quantification of ZO-1, occludin, and claudin 5 length on lectin+ endothelial profiles in these mice (D). Scale bar = 25 µm. Boxes in A–C are sites taken for higher magnification insets shown below single and merged images, scale bar = 10 µm. Mean ± SEM, n = 5 mice/group. (E and F) Immunoblotting for ZO-1, occludin, and claudin 5 in brain capillaries (E) and their relative abundance compared with β-actin (F) in Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice. Mean ± SEM, n = 3 mice/group. β-actin, loading control. (G) CypA, MMP9, and lectin+ endothelium (left: purple, CypA and lectin; white, MMP9 and lectin); CypA, lectin+ endothelium, and CD13+ pericytes (middle: purple, CypA and lectin), and MMP9, lectin+ endothelium, and CD13+ pericytes (right: purple, MMP9 and lectin) in brain capillaries from Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice. Scale bar = 10 µm. (H) Ppia and Mmp9 mRNA relative abundance normalized to Gapdh (housekeeping gene) in brain endothelial cells and pericytes from Lrp1lox/lox; Tie2-Cre and Lrp1lox/lox mice. Mean ± SEM, n = 5 isolates/group. (I) Gelatinase activity in brain endothelial cell medium from Lrp1lox/lox and Lrp1lox/lox; Tie2-Cre mice with or without si_Mmp9_ or si_Mmp2_. AU, arbitrary units. Mean ± SEM, n = 4 isolates/group. (J and K) Inhibition of Mmp9 mRNA (J) and activated MMP9 (K) by CypA silencing (si.Ppia), the CypA inhibitor cyclosporine A (CsA; 42 nM), and the NF-κB inhibitor PDTC (20 µM) in brain endothelial cells from Lrp1lox/lox; Tie2-Cre mice. (L–N) Inhibition of Ppia mRNA (L), Mmp9 mRNA (M), and active MMP9 (N) in brain endothelial cells from Lrp1lox/lox; Tie2-Cre mice after adenoviral LRP1 (Ad.m_LRP1_) reexpression in the presence of astrocyte-derived murine apoE (40 nM). Ad_GFP_, control. Mean ± SEM, n = 3 isolates/group. All mice were 2 mo old. In D and F, significance was determined by Student’s t test. In H–N, significance was determined by one-way ANOVA followed by Bonferroni post hoc test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure S4.

Figure S4.

Loss of collagen IV and hemosiderin deposits in Lrp1lox/lox; Tie2-Cre mice and additional characterization of reagents used in Fig. 4 . (A and B) Immunostaining for collagen IV (CollV; magenta) and fluorescent staining for lectin+ endothelium (green) in the cortex of 2-mo-old Lrp1lox/lox;Tie2-Cre and Lrp1lox/lox control mice (A), and quantification of collagen IV length on brain capillary (<6 µm in diameter) lectin+ endothelial profiles in these mice (B). Scale bar = 25 µm. Mean ± SEM, n = 5 mice/group. (C and D) Prussian blue hemosiderin deposits in 2-mo-old Lrp1lox/lox;Tie2-Cre mouse and lack of Prussian blue deposits in Lrp1lox/lox control (C), and quantification of Prussian blue+ hemosiderin deposits in these mice (D). Scale bar = 20 µm. Mean ± SEM, n = 3 mice/group. (E) Inhibition of Ppia mRNA (encoding CypA) by si_Ppia_, but not si_Control,_ in brain endothelial cells isolated from 2-mo-old Lrp1lox/lox; Tie2-Cre mice. (F) Representative immunoblotting of mLRP1 in brain endothelial cells from 2-mo-old Lrp1lox/lox; Tie2-Cre mice after adenoviral-mediated reexpression with Ad.m_LRP1_ but not control Ad.GFP virus. Significance by Student’s t test; ns, not significant; ***, P < 0.001; ****, P < 0.0001.

Figure S5.

Figure S5.

Short-term treatment with CypA inhibitor cyclosporine A reverses BBB breakdown in Lrp1lox/lox;Tie2-Cre mice. (A–C) CypA (top, red) and MMP9 (bottom, red) immunostaining in brain capillary (<6 µm in diameter) lectin+ endothelium (blue) in the cortex of 2-mo-old Lrp1lox/lox control and Lrp1lox/lox;Tie2-Cre mice treated for 7 d with cyclosporine A (see Materials and methods) or vehicle (A), and quantification of CypA (B) and MMP9 (C) fluorescent intensity in brain capillary lectin+ endothelium in these mice. Scale bar = 10 µm. Mean ± SEM, n = 6–7 mice/group. (D–F) IgG (top, purple) and fibrinogen (bottom, purple) immunostaining and lectin+ endothelial brain capillary (<6 µm in diameter) profiles (green) in the cortex of 2-mo-old Lrp1lox/lox control and Lrp1lox/lox;Tie2-Cre mice treated with cyclosporine A or vehicle (D), and quantification of IgG (E) and fibrinogen (F) perivascular capillary deposits in the cortex (Ctx) and hippocampus (Hp) of these mice. Scale bar = 20 µm. Mean ± SEM, n = 5–6 mice/group. (G) Quantification of ZO-1, occludin, and collagen IV length on lectin+ endothelial capillary profiles in the cortex of 2-mo-old Lrp1lox/lox;Tie2-Cre mice treated with cyclosporine A or vehicle compared with Lrp1lox/lox. Mean ± SEM, n = 5 mice/group. In B, C, and E–G, significance was determined by one-way ANOVA followed by Bonferroni post hoc test, *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Figure 5.

Figure 5.

Cyclophilin A inhibitor Debio-025 reverses vascular phenotype and protects from neurodegeneration in Lrp1lox/lox; Tie2-Cre mice. (A and B) MMP9 immunoreactivity in cortical endothelium (A) and quantification (B) in Lrp1lox/lox; Tie2-Cre mice after vehicle or Debio-025 treatment. Scale bar = 10 µm. Mean ± SEM, n = 10 mice/group. (C–E) IgG and fibrin deposits in the cortex (C) and quantification of their deposits in the cortex (Ctx) and hippocampus (Hp; D and E) in Lrp1lox/lox and Lrp1lox/lox; Tie2-Cre mice after vehicle or Debio-025 treatment. Scale bar = 20 µm. Mean ± SEM, n = 14 mice/group. (F–I) ZO-1 (F, purple), occludin (G, purple), and claudin 5 (H, purple) colocalization with endothelial lectin (green) in the cortex of 4-mo-old Lrp1lox/lox; Tie2-Cre mice treated with vehicle or Debio-25, and quantification of ZO-1, occludin, and claudin 5 length on lectin+ endothelial capillary profiles in these mice (I). Scale bar = 25 µm. Boxes in F–H are sites taken for higher magnification insets shown below single and merged images. Scale bar = 10 µm. White (merged), colocalization of ZO-1, occludin, or collagen with lectin. Mean ± SEM, n = 5 mice/group. (J and K) BBB Ktrans maps (J) and Ktrans values in the cortex and hippocampus (K) in Lrp1lox/lox; Tie2-Cre mice after vehicle or Debio-025 treatment, compared with Lrp1lox/lox mice treated with vehicle. Scale bar = 0.5 mm. Mean ± SEM, n = 5–6 mice/group. (L–N) NeuN+ neurons and SMI312+ neurites (L) and quantification of NeuN+ neurons and SMI312+ neurites in the cortex and hippocampus (M and N) in 4-mo-old Lrp1lox/lox and Lrp1lox/lox; Tie2-Cre mice after vehicle or Debio-025 treatment. Scale bar = 50 µm. Mean ± SEM, n = 14 mice/group. (O–R) Burrowing (O), nest construction (P), novel object location (Q), and recognition (R) in Lrp1lox/lox and Lrp1lox/lox; Tie2-Cre mice after vehicle or Debio-025 treatment. Mean ± SEM, n = 14 mice/group. All mice were 4 mo old. Debio-025 (10 mg/kg/d) or vehicle were given for 30 d starting at 3 mo of age. In B, significance was determined by Student’s t test. In D, E, I, K, and M–R, significance was determined by one-way ANOVA followed by Bonferroni post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

References

    1. Aleksinskaya, M.A., van Faassen E.E.H., Nelissen J., Janssen B.J.A., De Mey J.G.R., Hanemaaijer R., Rabelink T., and van Zonneveld A.J.. 2013. Identification of free nitric oxide radicals in rat bone marrow: implications for progenitor cell mobilization in hypertension. PLoS One. 8:e57761. 10.1371/journal.pone.0057761 -DOI -PMC -PubMed
    1. Barnes, S.R., Ng T.S.C., Santa-Maria N., Montagne A., Zlokovic B.V., and Jacobs R.E.. 2015. ROCKETSHIP: a flexible and modular software tool for the planning, processing and analysis of dynamic MRI studies. BMC Med. Imaging. 15:19. 10.1186/s12880-015-0062-3 -DOI -PMC -PubMed
    1. Bell, R.D., Winkler E.A., Sagare A.P., Singh I., LaRue B., Deane R., and Zlokovic B.V.. 2010. Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging. Neuron. 68:409–427. 10.1016/j.neuron.2010.09.043 -DOI -PMC -PubMed
    1. Bell, R.D., Winkler E.A., Singh I., Sagare A.P., Deane R., Wu Z., Holtzman D.M., Betsholtz C., Armulik A., Sallstrom J., et al. 2012. Apolipoprotein E controls cerebrovascular integrity via cyclophilin A. Nature. 485:512–516. 10.1038/nature11087 -DOI -PMC -PubMed
    1. Ben-Zvi, A., Lacoste B., Kur E., Andreone B.J., Mayshar Y., Yan H., and Gu C.. 2014. Mfsd2a is critical for the formation and function of the blood-brain barrier. Nature. 509:507–511. 10.1038/nature13324 -DOI -PMC -PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources