LRP1 is developmentally unregulated by OPCs.

<p>Coronal sections of E15.5 (a), E18 (c), P5 (e,i) and P60 (k,m) mouse brain were immunolabelled to detect OPCs (PDGFRα, green) and LRP1 (red). The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (b,d,f,h,j) secondary antibody alone controls. (k) Graphical representation of the percentage of OPCs that express LRP1 in the brain at each age. Data (n = 3 mice analyzed per age) were compared using a one-way ANOVA with a Bonferroni’s post-hoc test, and expressed as mean ± std. * P<0.05, **** P<0.0001. All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Scale bars represent 17μm. CC = corpus callosum, Ctx = cortex.</p

Microglia in the brain stably express LRP1 throughout life.

<p>Coronal sections of E13.5 (a), E15.5 (c), E18 (e), P5 (g, i) and P60 (k,m) mouse brain were immunolabelled to detect microglia (Iba1, green) and LRP1 (red). The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (o) Graphical depiction of the proportion of Iba1⁺ cells that expressed LRP1. Results were compared using a one-way ANOVA with a Bonferroni’s post-hoc test, expressed as means ± std and are representative of three independent experiments. (b, d, f, h, j, l, n) secondary antibody alone controls. All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Scale bars represent 17μm. Ctx = cortex, CC = corpus callosum.</p

LRP1 is highly expressed in the brain.

<p>Whole brain lysates from E13.5, P5 and P62 wildtype mice were analyzed by western blot to detect LRP1 (Fig 1A) and GAPDH (Fig 1A) protein expression. (c) Band pixel intensity was quantified, normalized to the loading control (GAPDH), and shows that LRP1 expression is significantly elevated in the postnatal brain compared to the embryonic (P = 0.001) and adult (P = 0.0004) brain. Results were compared using a one-way ANOVA with a Tukey’s post-hoc test, expressed as mean ± std. **P<0.01, ***P<0.001.</p

NeuN-positive neurons express LRP1, but parvalbumin-positive interneurons do not.

<p>Coronal sections through the P5 (a) and P60 (c) mouse brain, and transverse sections through the adult mouse spinal cord (e) were immunolabelled to detect mature neurons (NeuN, green) and LRP1 (red). g) Coronal section of an adult (P60) mouse brain immunolabelled to detect parvalbumin (green) and LRP1 (red). (b,d,f,h) secondary alone controls. The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Arrow heads represent parvalbumin⁺ neurons that do not express LRP1. Scale bars represent 17μm. Ctx = cortex, SC = spinal cord.</p

Newly formed oligodendrocytes do not express LRP1.

<p>Tamoxifen was administered to Pdgfra-CreER^T2::Rosa26-YFP transgenic mice at P57 to label OPCs and trace them as they generate new oligodendrocytes until P64. Single scan confocal images were collected through the corpus callosum (CC) following immunolabelling with YFP (green), LRP1 (red) and either PDGFRα or CC1 (blue). <b>a-a”‘</b> YFP⁺, PDGFRα⁺ cells were also LRP1⁺. <b>b-b”‘</b> YFP⁺, PDGFRα-negative cells were also negative for LRP1. <b>c-c”‘</b> YFP⁺ CC1⁺ cells were found to be LRP1-negative. White arrows indicate regions of co-localisation. Arrowheads indicate oligodendrocyte cell bodies, which do not express LRP1. Scale bars represent 17μm. CC = corpus callosum.</p

LRP1 is expressed by radial glia in the developing brain and spinal cord.

<p>Coronal sections of the E13.5 (a), E15.5 (c) and E18 (g)mouse brain and transverse sections of the E15.5 spinal cord (e) were immunolabelled to detect radial glia (RC2, green) and LRP1 (red). The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (b,d,f,h,j) secondary antibody alone controls. All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Scale bars represent 17μm. SC = spinal cord.</p

LRP1 is highly expressed by fibrous astrocytes.

<p>Coronal brain sections from P5 (c) and P60 (a,e,i) mice and transverse spinal cord sections from P60 mice (g) were immunolabelled to detect astrocytes (GFAP, green) and LRP1 (red). The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (b,d,f,h,j) secondary antibody alone controls. All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Scale bars represent 17μm. CC = corpus callosum, SC = spinal cord, Ctx = cortex, SVZ = subventricular zone and LV = lateral ventricle.</p

Microglia in the spinal cord express high levels of LRP1.

<p>Transverse sections of E15.5 (a) and P60 (c) spinal cord were immuno-labelled to detect microglia (Iba1, green) and LRP1 (red). The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (b,d) secondary antibody alone controls. All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Scale bars represent 17μm. SC = spinal cord.</p

Oligodendrocytes do not express LRP1.

<p>Coronal sections through the P60 mouse brain (a) and transverse sections through P60 mouse spinal cord (c) were immuno-labelled to detect oligodendrocytes (CC1, green) and LRP1 (red). The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (b,d) secondary antibody alone controls. All images are single z plane confocal scans. Arrowheads indicate oligodendrocyte cell bodies, which do not express LRP1. Scale bars represent 17μm. CC = corpus callosum, SC = spinal cord.</p

OPCs in the spinal cord express LRP1 in their cell bodies and processes.

<p>Transverse sections of E15.5 (a) and P60 (c) mouse spinal cord were immuno-labelled to detect OPCs (PDGFRα, green) and LRP1 (red). The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (b,d) secondary antibody alone controls. All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Scale bars represent 17μm. SC = spinal cord.</p