Biochemical identification of PGRN-interacting proteins.

<p>(<b>A</b>) Description of the experiment in <i>C</i>. mPGRN-HA stably transfected HEK293 cells were treated with the chemical crosslinker DSS. Immunoprecipitation was performed with an anti-HA antibody. The immunoisolates were analyzed by SDS-PAGE, which was then silver stained. Specific bands were cut out and analyzed by mass spectrometry. (<b>B</b>) Image of a gel after silver staining. The identities of bands 1–4 are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026454#pone-0026454-t001" target="_blank">Table 1</a>.</p

Biochemical identification of PGRN-interacting proteins.

<p>(<b>A</b>) mPGRN-AP or mPGRN-HA fusion protein constructs were stably transfected into HEK293 cells. Cell lysates or culture medium was analyzed on Western blot with anti-mPGRN antibody. This antibody specifically recognizes mPGRN but not the endogenous hPGRN in HEK293 cells. β-actin was used as the loading control. (<b>B</b>) Stably transfected HEK293 cells expressing mPGRN-HA were treated with cross linkers, followed by immunoprecipitation (IP) with HA antibody or control IgG. Immunoisolates were analyzed on Western blot with anti-mPGRN antibody. UT, untransfected HEK293 cells; ST, stably transfected cells.</p

Identification of proteins that are crossed-linked with progranulin.

<p>Identification of proteins that are crossed-linked with progranulin.</p

Expression of endogenous PGRN in different brain cell types.

<p>(<b>A–C</b>) mPGRN is expressed in MAP2-positive cultured mouse cortical neurons. (<b>D–F</b>) mPGRN is present in the processes of astrocytes in mixed brain cell cultures. (<b>G–I</b>) mPGRN seems to be localized in LAMP1-positive vesicles. (<b>J–L</b>) mPGRN is highly expressed in Iba1-positive cultured mouse microglia. (<b>M–O</b>) Anti-mPGRN specifically recognizes the endogenous mPGRN protein since the immunostaining signal is absence in Iba1-positive microglia (M) isolated from <i>GRN</i> knockout mice (N). Scale bar: 20 µm for all panels except G–I (3 µm).</p

Loss of ERp57 activity caused reduced PGRN secretion.

<p>(<b>A</b>) Western blot analysis of the efficiencies of siRNA knockdown of ERp57 expression in stably transfected HEK293 cells expressing mPGRN. β-tubulin was used as the loading control. (<b>B</b>) qRT-PCR analysis of relative mPGRN mRNA levels after siRNA treatment. (<b>C</b>) mPGRN levels in the culture medium were measured by Western blot with anti-mPGRN antibody. <i>Asterisk</i> indicates a protein band of unknown identity visualized by staining of the Western blot membrane with Ponceau S to indicate equal loading. (<b>D</b>) Quantification of mPGRN levels in <i>C</i>. The values are mean ± SEM. *<i>p</i><0.02, **<i>p</i><0.002 vs. scrambled control. This experiment was repeated three times with similar results. (<b>E</b>) A vector-based shRNA construct reduced ERp5 expression level in stably transfected HEK293 cells expressing mPGRN. β-tubulin was used as the loading control. (<b>F</b>) Levels of secreted mPGRN were reduced in the culture medium of HEK293 cells with partial knockdown of ERp5. <i>Asterisk</i> indicates a protein band of unknown identity visualized by staining of the western blot membrane to indicate equal loading. (<b>G</b>) Quantification of the levels of mPGRN in <i>F</i>. Values are mean ± SEM. *<i>p</i><0.02. This experiment was repeated three times with similar results.</p

Additional file 1: of The lysosomal protein cathepsin L is a progranulin protease

Figure S1. (a) Isolated lysosomes and total lysate from HEK293 cells treated with non-targeting (Con) or GRN transcript targeting siRNA were analyzed for PGRN-specific signal by western blot using the goat polyclonal PGRN antibody. (b) The same samples were also analyzed for GAPDH and lysosomal markers, Lamp2 and Cat D. Figure S2. Cat L (5 ng) efficiently processed PGRN (50 ng) into poly-granulin fragments in 1 h reaction time under pH 4.5. Under the same reaction condition, co-incubation with Cat L inhibitor, Z-FF-FMK blocked proteolytic processing of PGRN by Cat L in a dose-dependent manner. Figure S3. Annotated MS/MS spectra of the identified granulin peptides shown in Fig. 1d and in Tables 1 and 2. The Proteome Discoverer result files (.msf) were imported into the Scaffold 4.3 (Proteome Software) for sequence annotation. The peptides that were identified by both SEQUEST and Mascot above the cutoff values (SEQUEST: 1.3 for singly and doubly charged peptides, 2.5 for triply charged peptides; and Mascot Ion Score: 20) were manually evaluated PGRN peptides generated by Cat L activity were designated as CL-1 to CL-10; whereas the PGRN peptides generated by elastase activity were designated as EL-1 to EL-19. The fact that all the measured precursor masses of the identified peptides were within or around 1 ppm of the theoretical masses and that the tandem mass spectra (MS/MS) exhibit a continuous stretch of b- or y- ion series, or clear peak assignments, indicating confident identifications. Sequence assignments were further supported by multiple spectra of successive cleavages of the same sequence. (DOCX 6702 kb

Cellular stress decreases TDP-43 levels in patient neurons with the TDP-43 A90V mutation.

<p>(<b>A</b>) The level of full length TDP-43 in the insoluble fraction of patient neurons remains the same as control neurons without STS treatment but decreases with STS treatment. (<b>B</b>) Quantification of the experiment in Panel A. (<b>C</b>) TDP-43 level in the soluble fraction of patient neurons decreases after STS treatment. (<b>D</b>) Quantification of the experiment in Panel C. The relative level of TDP-43 in each lane is normalized with the tubulin band and quantification is based on three independent experiments. (<b>E</b>) Western blot analysis of the levels of endogenous (Endo) and transfected TDP-43 in cell lines stably expressing tetracycline (tet)-inducible either wildtype (WT) TDP-43-Flag or TDP-43 A90V-Flag with or without STS treatment. The expression of transfected TDP-43 WT or TDP-43 A90V is induced by tetracycline. The A90V mutation alone is insufficient to decrease TDP-43 level in this assay. (<b>F</b>) Quantification of the experiment in Panel E. There is no statistical difference between samples treated with and without STS.</p

Patient neurons with the TDP-43 mutation are more susceptible to staurosporine and display increased TDP-43 mislocalization under stress.

<p><b>(A)</b> Viability of control and patient neurons after exposure to10–100 nM staurosporine (STS) for 24 hours. More patient neurons die after treatment with 100 nM STS. Values are mean ± SEM. *: <i>p</i><0.05 (Student's t-test). <b>(B)</b> The percentages of TUJ1⁺ control and patient neurons with mislocalized TDP43 without any stressor treatment. In Panels A and B, iPSC lines 37L20, 37L25, 36L10 and 36L11 were used. <b>(C, D)</b> Representative images of TDP-43 and TUJ1 staining in control and patient neurons differentiated from iPSC lines 37L20 and 36L10 after treatment with 100 nM staurosporine for 24 hours (C) and the percentage of TUJ1⁺ control and patient neurons showing mislocalized TDP-43 (D). After STS treatment, more patient neurons show TDP-43 cytoplasmic mislocalization. Some cells are non-neuronal cells, for instance, some cells with large nuclei are not labeled by MAP2. Values are mean ± SEM. *: <i>p</i><0.05 (Student's t-test). <b>(E, F)</b> Representative images of FUS and TUJ1 staining in control and patient neurons differentiated from 37L20 and 36L11 after treatment with 100 nM staurosporine for 24 hours (E) and the percentage of TUJ1⁺ control and patient neurons differentiated from 37L20, 37L25, 36L10 and 36L11 showing mislocalized FUS (F). FUS localization is not affected by the <i>TARDBP</i> A90V mutation.</p

Generation and characterization of FTD/ALS patient-specific iPSC lines with the TDP-43 A90V mutation.

<p>(<b>A</b>) Sequencing of genomic DNA from patient fibroblasts confirmed the presence of the missense TDP43 A90V (c.269 C>T) mutation. (<b>B</b>) Schematic representation of the steps from the collection of patient fibroblasts to the derivation of postmitotic neurons. Fibroblasts, iPSCs and neurons were immunostained with TDP-43, NANOG and TUJ1 antibodies, respectively. (<b>C</b>) Quantitative RT-PCR analysis of the levels of total and endogenous expression of <i>OCT4</i>, <i>CMYC</i>, <i>SOX2</i>, and <i>KLF4</i> in iPSC and H9 ESC lines. Values were normalized to <i>GAPDH</i> levels. (<b>D</b>) No chromosomal abnormalities were found in any of the selected iPSC lines. The karyotypes of control line 37L20 and patient line 36L10 are shown.</p

Expression of miR-9 and its precursors is reduced in patient neurons under stress.

<p>(<b>A</b>) Relative expression levels of mature miR-9 in neurons derived from two control and three patient iPSC lines containing the A90V mutation with or without STS treatment (left panel). The averages in control and patient neurons are shown on the right. (<b>B</b>) Expression of pre-miR-9. (<b>C</b>) Expression of pri-miR-9-2. The average of different lines from the same individual is set as 1. The results in A–C were confirmed from additional two independent neuronal differentiation experiments. (<b>D</b>) The relative expression levels of pri-miR-9-2 and pre-miR-9-2 in neurons derived from two patient iPSC lines containing the <i>TARDBP</i> M337V mutation was reduced. This result is based on two independent differentiation experiments. (<b>E</b>) TDP-43 levels were greatly decreased in primary mouse neurons transfected with two <i>TARDBP</i> shRNAs. (<b>F</b>) Expression of pri-miR-9-2 is decreased in primary mouse neurons transfected with <i>TARDBP</i> shRNA. Values are mean ± SEM. **: <i>p</i><0.01 (Student's t-test).</p