The neuronal transcription factor MEIS2 is a calpain-2 protease target

Abstract

Tight control over transcription factor activity is necessary for a sensible balance between cellular proliferation and differentiation in the embryo and during tissue homeostasis by adult stem cells, but mechanistic details have remained incomplete. The homeodomain transcription factor MEIS2 is an important regulator of neurogenesis in the ventricular-subventricular zone (V-SVZ) adult stem cell niche in mice. We here identify MEIS2 as direct target of the intracellular protease calpain-2 (composed of the catalytic subunit CAPN2 and the regulatory subunit CAPNS1). Phosphorylation at conserved serine and/or threonine residues, or dimerization with PBX1, reduced the sensitivity of MEIS2 towards cleavage by calpain-2. In the adult V-SVZ, calpain-2 activity is high in stem and progenitor cells, but rapidly declines during neuronal differentiation, which is accompanied by increased stability of MEIS2 full-length protein. In accordance with this, blocking calpain-2 activity in stem and progenitor cells, or overexpression of a cleavage-insensitive form of MEIS2, increased the production of neurons, whereas overexpression of a catalytically active CAPN2 reduced it. Collectively, our results support a key role for calpain-2 in controlling the output of adult V-SVZ neural stem and progenitor cells through cleavage of the neuronal fate determinant MEIS2.

Keywords: Adult neurogenesis; Calpain; MEIS homeodomain protein; Stem cell; Subventricular zone; Transcription factor.

Fig. 1.

Calpain-2 protease activity is high in primary aNS and downregulated upon differentiation. (A) Schematic representation of the neurosphere assay. (B) Normalized transcript levels of calpain proteases in aNS and neurons. n=3. nd, no transcript detected. P-values are indicated [one-way ANOVA multiple comparison analysis (MCA; Tukey's post test)]. (C) Absolute calpain activity in whole-cell extracts of aNS cells compared to in vitro differentiated neurons expressed as relative fluorescence units (RFU). aNS n=4; neurons n=3. P-value is indicated (unpaired two-tailed t-test). (D) Calpain activity measurements (RFU) during in vitro differentiation of aNS. n=3. P-values are indicated (one-way ANOVA MCA, each timepoint compared to 0 h). (E) Representative immunoblots of CAPN2 protein during in vitro differentiation and densitometric analysis of n=3. Differences expressed as fold change (fc) relative to the 0 h value (set at 1). P-values are indicated (one sample t-test). All data are represented as mean±s.e.m.; n=biological replicates.

Fig. 2.

Inhibition of calpain-2 enhances whereas its activation reduces in vitro neuronal differentiation of aNS. (A) Schema of CAPN2 and CAPN4 domains. PC, protease domain; PEF, penta-EF-hand domain; FLAG, FLAG tag. Phosphosite S50 is indicated, the constitutively active mutation is S50D. (B) Calpain activity (RFU) in cell extracts of aNS transduced with Capn4 shRNA or non-targeting (NT) control shRNA. n=3. (C) Relative proportion of DCX+ neurons following transduction of Capn4 shRNAs expressed as fold change (fc) relative to NT shRNA control. n=3. (D) Representative images of DCX immunostaining (red) in cell transduced with Capn4 or NT shRNA visualized by GFP (green). White arrowheads mark double-positive cells; inserts, merged DCX, GFP and DAPI channels. Scale bars: 10 μm. (E) Calpain activity (RFU) in cell extracts of aNS transduced with CAPN2 or CAPN2^S50D followed by neuronally directed differentiation; n=3. (F) Relative proportion of DCX+ neurons following CAPN2^S50D transduction expressed as fc relative to transduction with empty vector (GFP); n=4. (G) Representative images of colocalization of DCX with GFP in cells transduced with the empty vector or co-localization of DCX with FLAG (green) in cells transduced with CAPN2^S50D fused to a FLAG-epitope. White arrowheads mark double-positive cells; inserts: merged DCX, FLAG and DAPI channels. Data are represented as mean±s.e.m. P-value is indicated (B, C, E and F; parametric one sample t-test). n=biological replicates. Scale bars: 10 μm.

Fig. 3.

MEIS2 in aNS and differentiating neurons. (A) Schematic overview of the antibody staining applied in this figure. Two antibodies, directed against the N- and C-terminus of MEIS2 have been used. Staining intensities of the N-terminal antibody and C-terminal antibody within single cells were analyzed and compared. (B) Scatter blot of single aNS fluorescence intensity values of cells immunostained with C- and N-terminal MEIS2-specific antibodies. Values are expressed as mean intensity values per cell nucleus; data points represent all cell nuclei assessed, from 230 cells. The few cells appearing shifted towards higher intensity values likely correspond to cells undergoing spontaneous differentiation. (C) Representative confocal images of analyzed cells counterstained with cytoplasmic marker phalloidin (Pha); dark blue arrow, undifferentiated cells weakly stained with N- and C-terminal MEIS2 antibody; light blue arrowhead, cell undergoing spontaneous differentiation displaying increased N- and C-terminal MEIS2 staining intensity. (D) Scatter blot of single cell fluorescence intensity values of cells stained with antibodies specific for C- or N-terminal MEIS2 or DCX, respectively, after 24 h of differentiation. Values are expressed as mean intensity values per cell nucleus; data points represent all cell nuclei assessed, from 980 cells. (E) Representative confocal images; dark blue arrow, immunoreactivity in a DCX− cell weakly stained with N- and C-terminal MEIS2 antibody; light blue arrowhead, immunoreactivity in a DCX+ cell showing increased N- and C-terminal MEIS2 staining intensity. (F) Mean intensity values per cell nucleus after automated quantification for N- and C-terminal MEIS2 antibody staining of the cells plotted in D, mean±s.e.m., n=F DCX+, 17 cells; DCX−, 963 cells; the cells were analyzed from eight samples in one experiment. P-values are indicated (Mann–Whitney U-test). Scale bars: 10 μm.

Fig. 4.

MEIS2 is a direct target of calpain-2. (A) Upper panel, MEIS2 protein domain structure. Conserved domains, MEINOX homology domain A (MHD-A), MEINOX homology domain B (MHD-B), nuclear export signal (NES), the homeodomain (HD) and nuclear localization signal (NLS) are marked. Lower panel, same scale graphical presentation of predicted calpain cleavage sites within MEIS2. Predictions were made using the GPS-CCD 2.0 tool. The y-axis represents the cleavage likelihood score (CLS) at respective amino acid positions. The red line at prediction score 0.654 indicates the maximum default cut-off value above which cleavage is assumed likely. (B) Schematic representation of the comparative calpain cleavage assays as assessed by immunoblotting. (C) Representative western blot of MEIS2–HA fragmentation following incubation with calpain-2 (solid gray box) or control without calpain-2 (dashed gray box). Immunoblot for HA epitope. Arrowhead, respective full-length proteins detected by HA-specific antibodies. Arrows, fragments detected upon incubation with calpain-2. Sizes of marker protein bands are indicated on the right. (D) Quantification of full-length MEIS2 signal on western blots following incubation with calpain-2 or control without calpain-2. For quantification, the area of full-length MEIS2 peak on optical density (OD) histogram of the respective lane was analyzed (mean±s.e.m.; n=5 biological replicates). Data are represented as mean±s.e.m. P-value is indicated (Wilcoxon one sample t-test). (E) Schematic representation of the experimental procedure for mass spectrometric identification of MEIS2 cleavage fragments. HA-tagged MEIS2 proteins were incubated with proteolytically active calpain-2, cleavage products were directly assessed by mass spectrometry. Blue, HA tag; red, MEIS2 portion of the fusion protein. (F) Upper panel: graphical representation of the MEIS2 protein domain structure. Lower panel: same scale graphical presentation of MEIS2 cleavage sites detected by mass spectrometry analysis (one experiment). The y-axis represents the frequency of cleavage sites (FCS) at the respective positions. Red asterisks mark sites referred to in the text. x-axis, MEIS2 amino acid positions.

Fig. 5.

Phosphorylation within the MHD-B proximal cluster reduces calpain-2-mediated cleavage of MEIS2. (A) Phosphorylated amino acids in MEIS2 identified by mass spectrometry, focusing on positions 190–220 (see also Fig. S3). Lines represent individual peptides that were detected, red spheres indicate phosphorylated residues. (B) Schematic presentation of MEIS2 protein domain structure. Conserved domains and phosphosites are indicated. (C) Phospho-mimetic MEIS2^4xPhos–HA construct generated by replacing S198, S204, T208 with aspartic acid and S206 with glutamic acid. (D) Representative immunoblots of MEIS2^4xPhos–HA following incubation with active calpain-2 (solid blue box) or control without calpain-2 (dashed blue box). Immunoblot for HA epitope. Arrowhead, respective full-length proteins detected by HA-specific antibodies. Sizes of marker protein bands are indicated on the right. (E) Quantification of the results in D (mean±s.e.m.; n=2). Quantification of full-length MEIS2 signal on western blot following incubation with calpain-2 or control without calpain-2. For quantification area of full-length MEIS2 peak on optical density (OD) histogram of the respective lane was analyzed. (F,G) Number of DCX+ neurons following overexpression of MEIS2-HA (F, n=5) or MEIS2^4xPhos-HA (G, n=3), expressed as fc relative to empty viral backbone control (GFP). Data are represented as mean±s.e.m. P-value is indicated (Wilcoxon one sample t-test). n=biological replicates.

Fig. 6.

Phosphorylation within the PEST-sequence enhances cleavage of MEIS2 by calpain. (A) Phosphorylated amino acids in MEIS2 identified by mass spectrometry, focusing on positions 250–270 (see also Fig. S3). Lines represent individual peptides that were detected; red spheres indicate phosphorylated residues. (B) Schematic presentation of MEIS2 protein domain structure. Conserved domains and the phosphosites at S261 and T264 are highlighted. (C) Partial amino acid sequence of MEIS2 showing S261, T264 and surrounding residues. Phospho-mimetic MEIS2^DD construct generated by replacing S261 and T264 with glutamic acid. Non-phospho-mimetic MEIS2^AI construct generated by replacing S261 by alanine and T264 by isoleucine. (D) Representative immunoblot images of MEIS2^DD–HA (blue, right) or MEIS2^AI–HA (red, left) after incubation with active calpain-2 (solid boxes) or control without calpain-2 (dashed boxes). Immunoblot for HA epitope. Arrowhead, respective full-length proteins detected by HA-specific antibodies. Sizes of marker protein bands are indicated on the right. (E,F) Quantification of full-length MEIS2 signal for the western blot shown in D following incubation with calpain-2 (solid bar) or control without calpain-2 (striped bar). For quantification area of full-length MEIS2 peak on optical density (OD) histogram of the respective lane was analyzed.

Fig. 7.

Dimerization with PBX1 reduces calpain-2-mediated cleavage of MEIS2. (A) Immunoblot images of calpain-2 cleavage assays of MEIS2-HA (upper-left panel) or co-purified (MEIS2–HA)–PBX1 dimers (upper-right panel), stained for HA. Lower panels: membrane re-probed for PBX1. Arrowhead, full-length MEIS2–HA. (B) Immunoblot of nuclear and cytoplasmic extracts isolated from aNS and serially developed for the antigens listed on the left. Immunoreactivity for CAPN2 was primarily detected in the cytosolic fraction. α-tubulin (aTUB) and lamin B1 (LMNB1) served are reference for the quality of subcellular fractionation. (C) A single confocal image of aNS transduced with Flag-tagged CAPN2 and stained with antibodies directed the Flag-epitope, MEIS2 and PBX1, counterstained with DAPI. Individual channels are shown in grayscale for clarity. MEIS2 immunoreactivity in the cytoplasm overlaps with CAPN2 but not with PBX1. Misexpression of CAPN2–Flag for detection was necessary because the CAPN2-specific antibody used in A does not perform well in fluorescent immunocytochemistry. Scale bar: 10 μm. Images in this figure representative of three repeats.

Fig. 8.

Schematic overview of MEIS2 post-translational regulation during aNS differentiation. In the adult V-SVZ, the intracellular protease calpain-2 displays high activity in stem and transient-amplifying progenitor cells (TAP), potentially mediated by transient Ca²⁺ signaling. MEIS2 is a direct target of calpain-2 in aNS. Phosphorylation within a conserved PEST sequence of MEIS2 increases MEIS2 susceptibility for calpain-2 cleavage resulting in MEIS2 fragmentation. The cells remain in an undifferentiated state and neurogenesis is ‘on hold’. During the process of neuronal differentiation, calpain-2 expression and activity decreases and phosphorylation of MEIS2 at conserved serine/threonine residues or the dimerization with PBX1 reduces the sensitivity of MEIS2 towards cleavage by calpain-2. MEIS2 is stabilized and contributes to olfactory bulb (OB) neurogenesis.