Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss

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Abstract

Pericytes are positioned between brain capillary endothelial cells, astrocytes and neurons. They degenerate in multiple neurological disorders. However, their role in the pathogenesis of these disorders remains debatable. Here we generate an inducible pericyte-specific Cre line and cross pericyte-specific Cre mice with iDTR mice carrying Cre-dependent human diphtheria toxin receptor. After pericyte ablation with diphtheria toxin, mice showed acute blood–brain barrier breakdown, severe loss of blood flow, and a rapid neuron loss that was associated with loss of pericyte-derived pleiotrophin (PTN), a neurotrophic growth factor. Intracerebroventricular PTN infusions prevented neuron loss in pericyte-ablated mice despite persistent circulatory changes. Silencing of pericyte-derived Ptn rendered neurons vulnerable to ischemic and excitotoxic injury. Our data demonstrate a rapid neurodegeneration cascade that links pericyte loss to acute circulatory collapse and loss of PTN neurotrophic support. These findings may have implications for the pathogenesis and treatment of neurological disorders that are associated with pericyte loss and/or neurovascular dysfunction.

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Fig. 1: Generation of a pericyte-specificc Cre line.
Fig. 2: Pericyte ablation with diphtheria toxin.
Fig. 3: Acute circulatory failure and rapid neuron loss after pericyte ablation.
Fig. 4: Loss of pericyte-derived pleiotrophin-mediated neuroprotection.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Code availability

We used our graphical user interface (GUI) code (https://github.com/petmri/ROCKETSHIP) running with MATLAB R2019a version (and anterior versions) for DCE MRI and DSC MRI analyses.

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Acknowledgements

The work of B.V.Z. is supported by the National Institutes of Health grants R01AG039452, R01NS100459, as well as R01AG023084, R01NS090904, R01NS034467, and the Foundation Leducq Transatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease reference no. 16 CVD 05.

Author information

A.M.N., A.M., K.K., Z.D. and Z.Z. designed and performed experiments and analyzed data. A.P.S., D.L., A.M., Z.D., M.D.S., P.K., Y.W., M.T.H., M.W., N.C.O., E.J.L. and X.X. performed experiments and analyzed data. A.M.N., K.K., A.M., Z.D. and M.D.S. contributed to the writing of sections of the manuscript. Z.Z and B.V.Z. designed all experiments, analyzed data and wrote the paper.

Correspondence to Berislav V. Zlokovic.

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Competing interest

The authors declare no competing interests.

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First co-authors: Angeliki M. Nikolakopoulou, Axel Montagne, Kassandra Kisler and Zhonghua Dai.

Senior co-authors: Zhen Zhao and Berislav V. Zlokovic.

Integrated supplementary information

Supplementary Figure 1 Two-promoter strategy for generating the pericyte-Cre mice.

(a,b) Immunoblotting of Cspg4-encoded transmembrane chondroitin sulfate proteoglycan NG2, PDGFRβ, and CD31 (a) and their relative abundance compared to β-Tubulin (b) in primary cultures of mouse oligodendrocyte precursor cells (OPCs), brain microvascular endothelial cells, pericytes, and vascular smooth muscle cells (VSMCs). Mean ± S.E.M. from 3 independent cultures; significance by one way ANOVA followed by Bonferroni posthoc test. (c,d) Confocal microscopy images showing abundant expression of PDGFRβ (c, green) and NG2 (d, green) proteins in mouse capillary (Caps) pericytes (arrowheads) compared to α-smooth muscle actin (SMA)+ (red) arterial (Art) VSMCs (arrows) in the isolated mouse retinal explants, representative of staining repeated independently in retinas from n = 3 mice with similar results. In c and d, Bar = 40 µm. (e) Venn diagram depicting NG2+ (red) and PDGFRβ+ (green) cell populations in the mouse central nervous system illustrates enriched NG2 and PDGFRβ expression in pericytes (the inner section, yellow) compared to OPCs or VSMCs. (f) Cultured primary mouse brain endothelial cells, pericytes and VSMCs co-transfected with Pdgfrβ-Flp and a Flippase-dependent reporter construct (pCMV:Frt-DsRed-Frt-EGFP) show GFP expression in pericytes and VSMCs, but not in endothelial cells. DsRed indicates transfected cells. (g) Cultured primary mouse brain endothelial cells, pericytes, and VSMCs co-transfected with Cspg4-FSF-CreER, Pdgfrβ-Flp and a Cre-dependent reporter (pCALNL-GFP) show that only pericytes, but not endothelial cells or VSMCs, successfully express GFP (green). mCherry (red), transfected cells. In f and g, representative images are from 3 independent experiments; Bar = 50µm. (Supplementary Figure 1). See Supplementary Figure 11 for full scans of Western blots used for quantification in a.

Supplementary Figure 2 Characterization of the pericyte-Cre; Ai14 mouse line.

(a) DNA gel images of PCR genotyping results from n = 7 F0 founders of the pericyte-CreER line, for Cre recombinase (JAX, generic Cre protocol), Flp recombinase (JAX, generic Flp protocol) and internal control using oIMR7338 and oIMR7339 primers (JAX). (b) western blot image representative of n = 3 repetitions with similar results showing expression of Flippase in brain microvessels from n = 4 pericyte-CreER mice (F1) and n = 1 littermate control (WT). Tubulin is used as loading control. (c) Representative confocal images, showing tdTomato expression in PDGFRβ+ pericytes. Bar = 20 µm. (d-h) Representative images showing that tdTomato is not expressed in Olig2+ oligodendrocytes (d), glial fibrillar acidic protein (GFAP)+ astrocytes (e,f; in f, high magnification of boxed region in e, and orthogonal view (right panel) showing that tdTomato+ pericyte is in close proximity, but not overlapping with the adjacent GFAP+ astrocytes), NeuN+ cortical neurons (g) and ionized calcium binding adaptor molecule-1 (Iba1)+ resident microglia (h). Bar = 20 µm. Representative images in c-h were obtained from the primary somatosensory cortex of n = 3 independent animals sacrificed two weeks after tamoxifen treatment (TAM, 7 consecutive injections, 40 mg/kg, i.p. daily) with similar results. (Supplementary Figure 1). See Supplementary Figure 11 for full scans of Western blots used for quantification in b.

Supplementary Figure 3 Astrocyte numbers, astrocytic endfeet aquaporin-positive coverage, microglia, and oligodendrocyte numbers in pericyte-CreER; iDTR mice treated with TAM and vehicle, or TAM and diphtheria toxin (DT).

(a-d) Representative confocal images and quantification of the number of GFAP+ astrocytes (a), astrocytic endfeet aquaporin-positive coverage of lectin-positive profiles (b), Iba1+ microglia (c) and Olig2+ oligodendrocytes (d) show no changes in TAM-treated pericyte-CreER; iDTR mice at 3 and 15 days post-DT compared to vehicle treatment. Mean ± S.E.M.; n = 3 mice/group; significance by one way ANOVA followed by Bonferroni posthoc test. Scale bar = 20 μm. (Supplementary Figure 2).

Supplementary Figure 4 Pericyte ablation leads to cerebral blood flow reductions and blood-brain barrier breakdown.

(a) Cerebral blood flow (CBF) maps in the primary somatosensory cortex generated from dynamic susceptibility-contrast (DSC)-MRI scans with gadolinium (Gd-DTPA) or iron oxide-based contrast agent (Ferumoxytol) in TAM-treated pericyte-CreER; iDTR mice at 3 days post-DT (red) or vehicle (blue) treatment. Mean ± S.D. n = 5 mice/group for Gd-DTPA and n = 6 mice/group for Gd-DTPA + DT; n = 3 mice/group for Ferumoxytol and Ferumoxytol + DT. One way ANOVA followed by Bonferroni posthoc test. (b,c) Confocal microscopy images in Ctx (b) and quantification in Ctx and hippocampus (Hipp) (c) of perivascular blood-derived fibrinogen deposits (red, b) in 3-month old TAM-treated pericyte-CreER; Ai14; iDTR mice at 0, 3, 6 and 9 days of DT or vehicle treatment, and 3 and 15 days post-DT or vehicle. In b, lectin+ endothelial profiles (blue); tdTomato+ pericytes (green). Bar = 20 µm. In c, mean ± S.E.M., n = 5 mice/group, significance by one way ANOVA followed by Bonferroni posthoc test. (d) Immunoglobulin G (IgG, red) leakage at 3 days post-DT or vehicle treatment representative of n = 3 independent mice/group with similar results in TAM-treated pericyte-CreER; iDTR; Ai14 mouse from a capillary lacking pericyte coverage (right) compared to no IgG leakage in a mouse with intact pericyte coverage treated with TAM and vehicle (left); tdTomato (green), lectin (white). Bar = 15 µm. (e-j) Confocal microscopy images in Ctx of tight junction proteins ZO-1 (e, red) and occludin (g, red) and adherens junction protein VE-cadherin (i, red) co-stained with lectin (blue), and quantification of ZO-1 (f), occludin (h), and VE-cadherin (j) length on lectin+ endothelial profiles in Ctx and Hipp in TAM-treated pericyte-CreER; iDTR mice at 3 days post-DT or vehicle treatment. Bar = 20 µm. Mean ± S.E.M., n = 5 mice/group. (k,l) Western blots of ZO-1, occludin, claudin-5 and VE-cadherin in brain capillaries isolated from TAM-treated pericyte-CreER; iDTR mice at 3 days post-DT or vehicle treatment (k), and quantification of their protein abundance relative to β-actin loading control (l). Mean ± S.E.M., n = 3 mice/group. (m,n) Electron microscopy micrographs showing capillaries in TAM-treated pericyte-CreER; iDTR mice at 15 days post-treatment with vehicle (m) or DT (n). Mice received retro-orbital injection of horse radish peroxidase (HRP; MW=44 kDa) and were sacrificed 2 hours later. Images are representative of 30 independent vessels from n = 3 mice per group with similar results that show the rarity of HRP-filled vesicles in the endothelium of either vehicle-treated (control) or DT-treated animal (red arrows). Higher magnification insets underneath illustrate tight junction (TJ) in vehicle-treated animal (m; magenta arrow) compared to loss of TJ proteins between endothelial cells of DT-treated animal (n, magenta arrowhead). Bar = 0.5 µm for m and n; bar = 0.2 µm for insets. In f, h, j, and l significance by two-tailed Student’s t-test. (Supplementary Figure 3a-i). See Supplementary Figure 11 for full scans of Western blots used for quantification in k.

Supplementary Figure 5 Pericytes ablation by diphtheria toxin does not lead to ‘rigor mortis’ of dying pericytes, obstruction of blood vessels with leukocytes and/or platelet-containing thrombi, but leads to development of vasogenic edema, reductions in red blood cells (RBC) capillary velocity, tissue hypoxia and elevated glutamate CSF levels.

(a) Time-lapse images of primary mouse pericytes isolated from Cre-inducible DTR transgenic mice (iDTR) and transduced with adeno-associated virus encoding Cre-recombinase (AAV-Cre) in which Cre-mediated excision of a STOP cassette renders cells sensitive to diphtheria toxin (DT). DTR+ pericytes were treated with vehicle, DT (0.1 µg/mL) or potassium chloride (KCl, 75 mM), and cell contractility and viability were assessed. Before incubation with vehicle, DT or KCl, pericytes were incubated (10 min) with Calcein-AM labeling live cells (live, green), and ethidium homodimer-1 labeling dead cells (dead, red); nuclei were labelled with cell-permeable Hoechst 33342 (blue). Time-lapse videos were taken immediately upon addition of vehicle, DT or KCl at 3 frames/minute for 45 mins. White dotted lines denote pericyte cell outline; Bar = 20 µm. Images are representative of 20 pericytes/group from three independent biological cultures with similar results. (b) Pericyte contractility was assessed by measuring the change in the length of the pericyte along the long axis of the cell, and quantified at 20 min, the time point at which the KCl positive control pericytes were fully contracted, and just prior to pericytes beginning to die in the DT-treated group. For the duration of the experiments, pericytes were incubated at 37 °C, 21% O2 and 5% CO2 in an incubation system for microscopy (KI4, Tokai Hit) and were imaged on a fluorescence microscope (BZ-9000, Keyence). Mean ± S.D.; n = 20 pericytes/group from three independent biological cultures. Significance by one way ANOVA followed by Bonferroni posthoc test. (c) Lectin+ endothelial vascular profiles (blue) and negative immunostaining for CD45+ leukocytes (red, top panel) and CD41+ platelets (red, bottom panel) in the cortical vessels of TAM-treated pericyte-CreER; iDTR mice at 3 days post-DT (left and middle panels). Positive control (right panel): accumulation of CD45+ leukocytes and CD41+ platelets in cortical blood vessels in the ischemic cortex of a mouse that underwent 1 h transient middle cerebral artery occlusion (MCAo)-induced stroke followed by 24 h reperfusion. Bar = 25 μm left panel; Bar = 50 μm middle and right (top panel); Bar = 10 μm middle and right (bottom panel). Representative images are from n = 6 mice for TAM-treated pericyte-CreER; iDTR mice at 3 days post-DT, and n = 3 mice for the MCAo experiment. (d-g) Vasogenic edema detected with longitudinal in vivo diffusion weighted MRI in primary somatosensory cortex (Ctx) and hippocampus (Hipp) of TAM-treated pericyte-CreER; iDTR mice at 9 days of treatment (DT or vehicle) and 15 days post-DT (red) compared to vehicle (blue). (d) Apparent coefficient diffusion (ADC) maps in vehicle-treated controls (top), after 9 days of DT (middle) and 15 days post-DT (bottom), representative of n = 9 vehicle and n = 4 DT-treated mice/group with similar results. Quantification of mean ADC values in Ctx (e) and Hipp (f); n = 9 vehicle and n = 4 DT-treated mice/group. Significance by one way ANOVA followed by Bonferroni posthoc test. (g) Increased cortical ADC values from the ischemic core region measured 15 days after MCAo-induced stroke in wild type mice (grey) compared to sham-operated controls; n = 4 mice/group; mean ± S.D. (h,i) Relationship between BBB permeability Ktrans constants and brain edema ADC coefficients in Ctx (h) and Hipp (i) of TAM-treated pericyte-CreER; iDTR mice at 9 days of treatment (DT or vehicle) and 15 days post-treatment (red, DT; blue, vehicle). The scatter plot diagrams include all animals at 9 days of DT and 15 days post-DT (n = 8) or vehicle (n = 10). Significance by two-tailed Pearson correlation; r2 = Pearson correlation coefficient. (j) Capillary kymographs from TAM-treated pericyte-CreER; iDTR mouse at 9 days of DT or vehicle measured by in vivo two-photon laser scanning microscopy, representative of 108 capillaries from n = 5 independent vehicle-treated mice, and 60 capillaries from n = 3 independent DT-treated mice/group with similar results. White lines provide a visual reference of the angles of the red blood cell (RBC) stripes compared to horizontal lines (dashed gray). (k) Quantification of capillary RBC velocities in TAM-treated pericyte-CreER; iDTR mice at 9 days of DT or vehicle. Averaged velocities from individual mice are shown. Number of capillaries evaluated per mouse are indicated on the graph in parentheses, in clockwise order from top left; a total of 108 capillaries from n = 5 vehicle-treated mice, and 60 capillaries from n = 3 DT-treated mice were evaluated; error is mean ± S.D. (l) Quantification of capillary diameter in TAM-treated pericyte-CreER; iDTR mice at 9 days of DT or vehicle. A total of 113 capillaries from n = 5 vehicle-treated mice, and 66 capillaries from n = 3 DT-treated mice were evaluated (21–24 capillaries/mouse); error is mean ± S.E.M. (m) Hypoxyprobe-1 (pimonidazole)+ hypoxic tissue (O2 <10 mm Hg) in the cortex and hippocampus of pericyte-CreER; iDTR mice at 9 days of DT compared to vehicle, representative of n = 4 independent vehicle-treated and n = 3 independent DT-treated mice/group with similar results. Bar = 50µm. (n) Quantification of hypoxyprobe-1+ area expressed as a percentage of total tissue area in the cortex. Mean ± S.E.M., n = 3 for DT treatment, n = 4 for vehicle treatment. (o) Cerebrospinal fluid glutamate levels in TAM-treated pericyte-CreER; iDTR mice at 9 days of DT or vehicle treatment, and in stroked mice 24 h after MCAo compared to sham-operated controls. Mean ± S.E.M.; n = 3 mice/group. In g, k, l n, o, significance by two-tailed Student’s t-test. (Supplementary Figure 3a-i).

Supplementary Figure 6 Limited Cre recombinase activity in peripheral organs, systemic physiologic parameters and liver and kidney analyses, and visual behavior testing after pericyte ablation with DT.

(a-d) Representative confocal microscopy images from 3 pericyte-CreER; Ai14 mice 14 days after TAM treatment (40 mg/kg per day for 7 days as in main figures) showing limited tdTomato expression in kidney (a), liver (b), heart (c) and skeletal muscle (d). PDGFRβ is used as pericyte marker, Lectin is used to label endothelium, and nuclei were stained with DAPI. Images in a-d representative of n = 3 independent mice with similar results. Bar = 50 µm. (e) Heart rate, respiratory rate (n = 4 vehicle-treated, n = 5 DT-treated mice/group), arterial pO2, pCO2, pH (n = 5 vehicle-treated, n = 6 DT-treated mice/group), and glucose levels (n = 4 vehicle-treated, n = 3 DT-treated mice/group); (f), liver analyses: alkaline phosphatase, ALP; alanine aminotransferase, ALT; aspartate aminotransferase, AST; creatine phosphokinase, CPK; albumin, total protein, total bilirubin. n = 4 vehicle-treated, n = 3 DT-treated mice/group. (g) kidney analyses: blood urea nitrogen, creatinine, sodium, calcium (n = 4 vehicle-treated, n = 3 DT-treated mice/group), and potassium (n = 5 mice/group) were not altered in TAM-treated pericyte-CreER; iDTR mice at 3 days post-DT or vehicle treatment. Mean ± S.E.M., Significance by two-tailed Student’s t-test. (Supplementary Figure 1, 2 and 3). (h) Visual behavior testing. TAM-treated pericyte-CreER; iDTR mice treated with DT (developing pericyte ablation) or vehicle (controls) underwent visual behavior testing at 15 days post-DT using a visual cliff test. Briefly, a transparent Plexiglass square arena was divided into two equal parts by aligning the middle of the arena with the edge of a table. The side sitting on the tabletop was considered a “shallow” side and the other one that is positioned over the floor area (50 cm high) a “deep” side. A patterned floor consisting of 3 cm black and white checked paper was placed bellow the arenas: on the shallow side the checked paper was placed immediately below the Plexiglass surface, while on the deep side the paper was placed on the floor creating an illusion of a cliff. Each animal was placed on the shallow side and the total time the animal spent exploring each side of the arena was recorded within a 5 min trial, and the percentage time spent in the shallow side was analyzed. As a positive control, blind C3H/HeJ mice were also tested. Circles represent percent time spent on shallow side of arena for each mouse tested; boxes represent mean ± S.D.. n = 8 C3H/HeJ control mice, n = 6 vehicle-treated, and n = 3 DT-treated pericyte-CreER; iDTR mice; one way ANOVA followed by Bonferroni posthoc test.

Supplementary Figure 7 Control studies showing that diphtheria toxin (DT) does not alter pericyte coverage, cerebral blood flow response, cerebrovascular integrity, neuronal numbers, neurofilament density, and behavior in iDTR mice treated with TAM and DT, or in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle.

(a,b) Confocal microscopy images (a) and quantification (b) of CD13+ pericyte coverage in the primary somatosensory cortex (Ctx) in iDTR animals treated with tamoxifen (TAM, 40 mg/kg i.p. daily for 7 consecutive days) and DT (0.1 µg per day for 10 consecutive days), and in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle, using the same protocol as in the main Figs. 2 and 3. The analyses were performed 3 days post-DT or vehicle treatment. Bar = 20 μm. Mean ± S.E.M.; n = 5 mice/group. (c) Cerebral blood flow (CBF) response to an electrical hindlimb stimulus by laser doppler flowmetry in iDTR animals treated with TAM + DT, and in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle 3 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group; one way ANOVA followed by Bonferroni posthoc test. (d,e) Confocal images (d; Bar = 20 μm) and quantification (e) of extravascular fibrinogen and IgG deposits in the primary somatosensory cortex of iDTR animals treated with TAM + DT, and Cre mice treated with TAM and vehicle at 3 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group. (f,g) Confocal images (f; Bar = 20 μm) and quantification of ZO-1 tight junction protein length (g) in the Ctx of iDTR animals treated with TAM + DT, and Cre mice treated with TAM and vehicle 3 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group. (h-j) Confocal images (h) and quantification of NeuN+ neurons (i) and SMI312+ neurofilaments (j) in the Ctx and Hipp of iDTR animals treated with TAM + DT, and Cre mice treated with TAM and vehicle 15 days post-DT or vehicle treatment. Bar = 50 μm. Mean ± S.E.M.; n = 5 mice/group. (k,l) Novel object location (k) and fear conditioning (l) in iDTR animals treated with TAM + DT, and in pericyte-CreER; iDTR mice (Cre) treated with TAM and vehicle at 15 days post-DT or vehicle treatment. Mean ± S.E.M.; n = 5 mice/group. In b, e, g, i-l, significance by two-tailed Student’s t-test. (Supplementary Figure 3 and Fig. 4e-k).

Supplementary Figure 8 Pleiotrophin expression in mouse pericytes and pleiotrophin (PTN)-mediated neuroprotection.

(a-c) Dual fluorescence in situ hybridization for Ptn mRNA and immunostaining of NeuN+ neurons (a), GFAP+ astrocytes (b), and olig2+ oligodendrocytes (c) in cortex; Bar = 10µm. (d) Dual fluorescence in situ hybridization for Ptn mRNA and immunostaining of CD13+ pericytes in isolated mouse brain microvessels; Bar = 20 µm. Images in a-d are representative from n = 3 independent mice with similar results. (e) Immunoblotting for PTN in primary cultured mouse brain pericytes, endothelial cells, oligodendrocyte precursor cells (OPCs), neurons, VSMCs, and astrocytes, and PTN expression levels relative to β-actin. Mean ± S.E.M., n = 3 independent cultures. Significance by one way ANOVA followed by Bonferroni posthoc test. (f,g) Immunostaining of the primary mouse cortical (Ctx) and hippocampal (Hipp) neurons for microtubule-associated protein 2 (MAP2) and nuclear DAPI staining. Neurons were cultured for 10 days with B27 neuronal growth supplement followed by B27 withdrawal (-B27) for 48 h in the presence and absence of pericyte-conditioned media containing pericyte-secreted mouse PTN at a concentration diluted to match mouse CSF levels with and without mouse PTN neutralizing antibody (anti-PTN; 500 ng/ml) or non-immune IgG (NI IgG), and/or mouse recombinant PTN (3.5 nM) (f); quantification of MAP2+ DAPI+ Ctx and Hipp neurons under experimental conditions as shown in f (g); Mean ± S.E.M., n = 3 independent cultures in triplicate. (h-k) Oxygen glucose deprivation (1% O2, no glucose)-induced (h,i) and glutamate (10 µM)-induced (j,k) neuronal cell death in the absence and presence of pericyte-conditioned media containing pericyte-secreted mouse PTN at a concentration concentrated to match mouse CSF levels with and without mouse PTN neutralizing antibody (anti-PTN; 500 ng/ml) or NI IgG, and/or mouse recombinant PTN (3.5 nM). Quantification as in g. Mean ± S.E.M., n = 3 independent cultures in triplicate. In g, i and k, significance by one way ANOVA followed by Bonferroni posthoc test for multiple comparisons. (Supplementary Figure 4). See Supplementary Figure 11 for full scans of Western blots used for quantification in e.

Supplementary Figure 9 PTN intracerebroventricular (ICV) infusions do not influence cerebrovascular changes after pericyte ablation.

(a-c) Confocal microscopy images (a; representative of n = 5 independent mice/group with similar results, Bar = 20 μm) and quantification of CD13+ pericyte coverage of lectin+ endothelial profiles on capillaries (<6 µm in diameter) in the cortex (Ctx) (b) and hippocampus (Hipp) (c) of TAM-treated pericyte-CreER; iDTR mice 15 days post-DT after ICV infusions with mouse recombinant PTN (3 nM, final CSF concentration) or aCSF (control PTN-free), and 2 weeks post-siPtn or siControl (scrambled siRNA) ICV treatment of TAM and vehicle-treated pericyte-CreER; iDTR mice. Mean ± S.E.M., n = 5 mice/group. (d-f) Cerebral blood flow (CBF) DSC-MRI maps (d) representative of n = 6 independent mice/group with similar results, and quantification in Ctx (e) and Hipp (f) of TAM-treated pericyte-CreER; iDTR mice at 15 days post-DT after ICV infusion with either PTN (3 nM, final CSF concentration) or control aCSF. Mean ± S.E.M., n = 6 mice/group. (g-i) BBB Ktrans DCE-MRI maps (g) representative of n = 5 independent aCSF-treated and n = 6 independent PTN-treated mice/group with similar results, and quantification in Ctx (h) and Hipp (i) of TAM-treated pericyte-CreER; iDTR mice at 15 days post-DT after ICV infusion with either PTN (3 nM, final CSF concentration) or control aCSF. Mean ± S.E.M., n = 5 aCSF-treated, n = 6 PTN-treated mice/group. (j-l) Confocal images in Ctx (j) representative of n = 5 independent mice/group with similar results, and quantification in Ctx (k) and Hipp (l) of immunoglobulin G (IgG) (red, j) deposits in TAM-treated pericyte-CreER; Ai14; iDTR mice at 15 days post-DT after ICV infusion with either PTN (3 nM, final CSF concentration) or control aCSF, and 2 weeks post-siPtn or siControl ICV treatment of TAM and vehicle-treated pericyte-CreER; iDTR mice. Blue, lectin+ endothelial profiles. Bar = 20 μm. Mean ± S.E.M., n = 5 mice/group. In b-c, e-f, h-i, k-l, significance by one way ANOVA followed by Bonferroni’s posthoc test. (Supplementary Figure 4).

Supplementary Figure 10 Depletion of PTN in pericytes by Ptn knockdown.

(a) Dual fluorescence in situ hybridization of Ptn mRNA and immunostaining for CD13+ pericytes in TAM-treated pericyte-CreER mice after 2 weeks post-siPtn or siControl ICV treatment. Images are representative of n = 3 independent mice/group with similar results. (b,c) Immunoblotting of PTN in brain capillaries (b) and cerebrospinal fluid (CSF) PTN levels (c) in TAM-treated pericyte-CreER mice after 2 weeks post-siPtn or siControl (scrambled siRNA) ICV treatment. Mean ± S.E.M.; n = 3 mice/group. In b, GAPDH, loading control. In b and c, significance by two-tailed Student’s t-test. (Supplementary Figure 4). See Supplementary Figure 11 for full scans of Western blots used for quantification in b.

Supplementary Figure 11 Full scans of all Western blots used for quantification in main Figs. 2c and 4c, and Supplementary Figures 1a, 2b, 4k, 8e, and 10b.

Dotted boxes indicate lanes presented as representative blots in the respective main and supplementary figures.

Supplementary information

Supplementary Figures 1–11 and Supplementary Table 1.

Reporting Summary

Supplementary Video 1

Time-lapse live imaging of mouse pericytes expressing human DTR after treatment with vehicle. Pericytes were incubated for 10 min with Calcein-AM, labeling live cells (green), and ethidium homodimer-1 labeling dead cells (red); nuclei were labelled with cell-permeable Hoechst 33342 (blue). Time-lapse videos were taken immediately upon addition of vehicle, at 3 frames/minute for 45 mins. Vehicle treatment did not result in pericyte cell death or contraction. Video is representative of 20 pericytes/group from three independent biological cultures.

Supplementary Video 2

Time-lapse live imaging of mouse pericytes expressing human DTR after treatment with diphtheria toxin (DT; 0.1 µg/mL). Pericytes were incubated for 10 min with Calcein-AM, labeling live cells (green), and ethidium homodimer-1 labeling dead cells (red); nuclei were labelled with cell-permeable Hoechst 33342 (blue). Time-lapse videos were taken immediately upon addition of DT, at 3 frames/minute for 45 mins. DT led to pericyte cell death, but did not have effect on pericyte contractility. Arrow points to pericyte undergoing cell death. Video is representative of 20 pericytes/group from three independent biological cultures.

Supplementary Video 3

Time-lapse live imaging of mouse pericytes expressing human DTR after treatment with 75 mM KCl. Pericytes were incubated for 10 min with Calcein-AM, labeling live cells (green), and ethidium homodimer-1 labeling dead cells (red); nuclei were labelled with cell-permeable Hoechst 33342 (blue). Time-lapse videos were taken immediately upon addition of KCl, at 3 frames/minute for 45 mins. KCl treatment led to pericyte contraction, but not cell death. Arrow points to pericyte undergoing contraction. Video is representative of 20 pericytes/group from three independent biological cultures.

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Nikolakopoulou, A.M., Montagne, A., Kisler, K. et al. Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss. Nat Neurosci 22, 1089–1098 (2019) doi:10.1038/s41593-019-0434-z

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