Supplemental Figure S1. PGRN Binding to Sortilin.

Transcription

1 1 Neuron, volume 68 Supplemental Data Sortilin-Mediated Endocytosis Determines Levels of the Frontotemporal Dementia Protein, Progranulin Fenghua Hu, Thihan Padukkavidana, Christian B. Vægter, Owen A. Brady, Yanqiu Zheng, Ian R. Mackenzie, Howard H. Feldman, Anders Nykjaer, and Stephen M. Strittmatter Supplemental Figure S1. PGRN Binding to Sortilin. (A) Size Exclusion Chromatography of recombinant PGRN protein. The indicated proteins, AP-PGRN, AP-PGRN-E, and mcherry-pgrn, were prepared from conditioned medium and then fractionated by size exclusion chromatography. The elution of the two AP proteins was monitored by AP activity with pnpp as substrate, and

2 the elution of the mcherry protein by fluorescence. The majority of protein species elutes at a position corresponding to the predicted size of a monomer. (B) AP-PGRN binding to neurons is displaced by untagged ligand. Cultured E17 Mouse cortical neurons at 21DIV were incubated with 10 nm AP-PGRN alone or 10 nm AP- PGRN plus 200 nm of His-PGRN at 4 C for 1 hour. The non-specific binding in the presence of His-PGRN is 18% of total binding. Scale bar = 50 μm. (C) Selectivity of PGRN for Sortilin as binding site. COS-7 cells expressing Sortilin, SorLA, NTR1 or NTR2 were incubated with conditioned media containing AP-fusion proteins as indicated. Binding was visualized with AP substrates. To confirm expression of SorLA, cells expressing Sortilin or SorLA were stained with anti-sortilin or anti-sorla antibodies. The expression of SorLA was further confirmed by immunoblot. (D-E) AP-GRN-E binds with high affinity to cortical neurons. (D) The binding of AP PGRN-E binding to cortical neurons 21 DIV is measured as a function of ligand concentration. Data are mean + sem. (E) The data from D are replotted. The K D is mean + sem from 4 independent experiments. 2

3 3 Supplemental Figure S2. Lack of PGRN Regulation of Neurite Outgrowth and Cell Survival in Cortical Culture. (A-C) Rat E18 primary cerebral cortex or hippocampal neurons were cultured with or without 300 nm Flag-PGRN for 24 hours and then stained with anti-ßiii tubulin to

4 visualize neurons and their neurites as shown in (C). The number of cells/well (B) and the neurite outgrowth per cell (A) was measured using an automated high content cell imager and neurite outgrowth analysis (ImageExpress, Molecular Dynamics). The data are mean + sd from 4 wells of an experiment representative of 3 independent cultures with similar results. In each well, more than 200 neurons were analyzed. No significant effect of PGRN was detected. (D, E) Rat E18 cerebral cortical cultures were exposed to standard medium or 150 nm Flag-PGRN or 500 nm staurosporine (positive control) for 7 days from 14 DIV to 21 DIV. Cultures were then fixed and stained for ßIII tubulin (green), nuclei (DAPI, blue) or cleaved caspase 3 (red). The percentage of cells positive for the apoptotic marker, cleaved caspase 3, was determined by automated image analysis. The data are mean + sd from 4 wells of an experiment representative of 3 independent cultures with similar results. In each well, more than 2,000 neurons were analyzed. No significant effect of PGRN was detected. 4

5 5 Supplemental Figure S3. Cellular Localization of PGRN and Sortilin in Spinal Cord Ventral Horn. (A) PGRN antibody is specific. Immunohistochemistry for PGRN was conducted for transverse L5 spinal cord sections form WT or GRN -/- mice that underwent sciatic nerve injury (resection) 21 days previously. No staining was seen in the GRN -/- samples. Scale bar, 50 μm. (B) Immunohistochemistry for PGRN, GFAP and Iba1 was conducted on L5 spinal cords of mice that had sciatic nerve injury (resection) 7 days previously. While colocalization of the microglial marker (Iba1) and injury-induced PGRN is observed, there is no co-localization of reactive astrocytes (GFAP) with PGRN. Scale bar, 50 μm. (C) Immunohistochemistry for Sortilin, NeuN and Iba1 was conducted on L5 spinal cords of mice that had sciatic nerve resection 7 days earlier. Colocalization is seen between Sortilin and NeuN. No colocalization was observed for Iba1 and Sortilin. Scale bar, 50 µm.

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7 7 Supplemental Figure S4. Lack of PGRN or Sortilin Degenerative Phenotype in MN after Axotomy Despite TDP-43 Redistribution. (A) One week after unilateral sciatic nerve injury, mice were sacrificed and tissue processed. Transverse sections containing the L5 ventral horn of injured or uninjured side was stained with anti-tdp-43 and anti-neun, as indicated. Note the increased cytoplasmic TDP-43 in the large MN cell bodies after axonal injury. Scale bar, 20 μm. (B, C) Three weeks after unilateral sciatic nerve injury, cross-sections of the L5 ventral root were processed. Intact axonal profiles were scored for each entire root. Data are the mean + sem for 4-6 roots per group. No significant differences were noted. Scale bar, 10 μm.

8 8 Supplemental Figure S5. Expression patterns for PGRN and Sortilin. Adult mouse expression data for GRN (A-C) and Sort1 (D-F) were downloaded from online databases. The microarray expression data (A, D) are from Gene Atlas (Su et al.,

9 2004; Wu et al., 2009) ( and the in situ hybridization analysis (B, C, E, F) from the Allen Brain Atlas (Lein et al., 2007) ( The in situ hybridization data are shown both in brightfield (B, E) and expression (C, F) views. Note high expression of Sort1 in multiple brain regions, and high expression of GRN in microglia. 9

10 10 Supplemental Figure S6. PGRN Levels in Sortilin and PGRN mutant mice. (A) Glycosylation of PGRN protein in the brain lysate. Tissue lysates collected from the cortex of 7-month-old mice of the indicated genotypes were subjected to PNGaseF treatment, Endo- -N-Acetylgalactosaminidase treatment or no enzyme treatment for 1 hour. The protein was then methanol precipitated, subjected to SDS-PAGE and

11 immunoblotted for PGRN. Both 73KDa and 78KDa PGRN protein bands collapse to one 63KDa band after PNGaseF treatment and do not do so with Endo- -N- Acetylgalactosaminidase treatment or no enzyme control. (B) Cerebral cortex lysates from 2-month-old mice were obtained from the indicated genotypes and immunoblotted for PGRN. (C) Quantification of PGRN level in the brain normalized to GAPDH level. For all genotypes, n = 4 mice. Mean + sem, **, P < 0.01, one-way ANOVA. (D) Cerebral cortex lysates from adult WT and GRN-/- mice were immunoblotted for Sortilin with an antibody recognizing the C-terminal region. The bottom panel shows an over-exposed view and the absence of any detectable carboxyl 15 kda fragment. (E) Cerebral cortex lysates from adult WT and Sort1-/- mice were immunoblotted for prosaposin (SAP), another Sortilin ligand. The two SAP panels were generated with different antibodies. While PGRN levels are increased in the Sort1-/- mice there is no change in prosaposin levels. (F) GRN mrna levels are not altered in mice lacking Sort1. RNA from cortex of 7- month-old mice was subjected to qrt-pcr with 2 different GRN probes. The resulting relative quantity of RNA was normalized to GAPDH mrna. No change in GRN mrna was seen in Sort1-/- compared to WT control. Mean + sem. 11