EMBO/soda-nel huggingface dataset:
Choroid Plexus-derived miR-204 Regulates the Number 
of Quiescent Neural Stem Cells in the Adult Brain (2019)
Figure 1

Figure 1. A subset of qNSCs (LRCs) expresses mRNA but not the protein for neurogenic fate determinants.

(A) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ.

(B) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells. Note that Meis2 mRNA positive LRCs have low (no) MEIS2 protein.

(C, D) IHC labeling for MEIS2 (C) and MCM6 (D) of LRCs positive for BrdU only and neuroblasts marked by DCX.

(E) Dot plot showing the proportion of BrdU+ LRCs negative for MEIS2 or MCM6 protein.

(F) Dot plot depicting the expression of miR-204 in prospectively isolated cells of neural lineage.

Data information: See also Fig. EV1. All fluorescent images are full Z-projections of confocal Z-stack. Boxed areas correspond to the higher magnification images in adjacent panels (C', C´, D' and D´); numbers(1-6) label the position of the cells zoomed in the adjacent panels (C', C´, D' D´). Abbreviations: qNSC, quiescent neural stem cell; aNSC, activated neural stem cell; NB, neuroblast; DE, diencephalon astrocytes; LRC, label retaining cell; SEZ, subependymal zone; LV, lateral ventricle. Data are shown as mean+/-SEM; each single dot represents independent biological replicate; significance was tested by non-parametric Kruskal-Wallis ANOVA; *-p value <0.05, **-p value <0.01. Scale bars (C, D) 50 µm, (C', C´, D', D´), 10 µm (B, numbered magnifications) 5 µm.
Choroid Plexus-derived miR-204 Regulates the Number 
of Quiescent Neural Stem Cells in the Adult Brain (2019)
Figure 2

Figure 2. miR-204 regulates neurogenic priming of adult NSCs.

(A) Schematic representation of the experimental workflow for labelling LRCs with BrdU in the adult SEZ and miR-204 inhibition using antagomirs.

(B) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi.

(C, D) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF (control, C) or AntimiR204 (D). White arrows indicate LRCs without detectable MEIS2 protein. The white arrowheads are pointing out the loss of neurogenic priming (LRCs positive for MEIS2 protein) upon the antagomir treatments.

Data information: See also Fig. EV1. All fluorescent images are full Z-projections of confocal Z-stack, orthogonal projections are single plane pictures. Boxed areas correspond to the higher magnification images in adjacent panels (C', D',) or orthogonal projections (C'', C´´, D''). Abbreviations: AntimiR, antagomir; aCSF, artificial cerebrospinal fluid; CTR, control; SEZ, subependymal zone; LV, lateral ventricle; WT, wildtype; dpi, days post injection, LRC, label retaining cell, OB, olfactory bulb; CTX, cortex; RMS, rostral migratory stream. Data are shown as mean+/-SEM; each symbol represents independent biological replicate; significance was tested by non-parametric Kruskal-Wallis ANOVA; *-p value <0.05. Scale bars (C, D) 50 µm, (C', D') 10 µm, (C'', C´´, D'') 5 µm.
Choroid Plexus-derived miR-204 Regulates the Number 
of Quiescent Neural Stem Cells in the Adult Brain (2019)
Figure 3

Figure 3. Loss of the neurogenic priming in vivo induces pre-mature differentiation of adult NSCs.

(A, B) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF (control, A) or AntimiR204 (B). Boxed areas correspond to higher magnifications in adjacent panels (A', B').

(C-E) Dot plots depicting the abundance of LRCs (BrdU+, Ki67-, DCX-), aNSCs and TAPs (BrdU-, Ki67+, DCX-) and neuroblasts (DCX+ Ki67+ or DCX+ Ki67-) in the SEZ 7 days after aCSF or AntimiR204 injection. Note that the number of LRCs (white arrows in A and B) is reduced in Antimir204 injected SEZ, while the number of NBs is increased.

(F, G) Micrographs depicting the elbow - the most ventral region of the RMS (F) and OB neuronal layers (G) upon injection of artificial CSF (control) or AntimiR204.

(H) Dot plot showing the density of BrdU+ cells (progeny of NSCs) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection.

Data information: See also Fig. EV2. All fluorescent images are full Z-projections of confocal Z-stack, (A' and B') are orthogonal projections through the Z-stack. Abbreviations: LRC, label retaining cell, TAPs, transit amplifying progenitors; SEZ, subependymal zone; LV, lateral ventricle; WT, wildtype; qNSC, quiescent neural stem cell; aNSC, activated neural stem cell; RMS, rostral migratory stream, OB, olfactory bulb;CTR, control. Data are shown as mean+/-SEM; each symbol represents independent biological replicate; significance was tested by non-parametric Kruskal-Wallis ANOVA; *-p value <0.05, **-p value <0.01, ***-p value <0.001. Scale bars (A, A', B, B') 20 µm, (F, G) 50 µm.
Choroid Plexus-derived miR-204 Regulates the Number 
of Quiescent Neural Stem Cells in the Adult Brain (2019)
Figure 4

Figure 4. ChP is a main source of the miR-204 that is released into the CSF.

(A) Micrographs of in situ hybridization for miR-204 in an adult mouse brain section. (A' and A'') are magnifications of boxed areas in (A or A'), respectively.

(B) Dot plot showing miR-204 levels in ChP and SEZ in the adult mouse brain measured by RT-qPCR.

(C) Agarose gel of RT-qPCR product loaded after the saturation phase showing presence of miR-204 and U6 in mouse ChP, SEZ and CSF.

(D, E) Plots depicting the number and size of EVs isolated from human CSF (D) and miR-204 levels (E) in EVs and EV-free CSF (n=2).

(F) Agarose gel of RT-qPCR analysis depicting levels of miR-204 in the EV-free supernatant and EV containing fraction of human CSF loaded at the saturation phase.

(G) Micrographs of transmission electron microscopy imaged extracellular vesicles (EVs) isolated from sham treated (upper row) and GW4869 treated animals (lower row). (G'' and G''') are magnifications of boxed areas in overview images to the left.

(H) Dot plot depicting miR-204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor.

Data information: Abbreviations: STR, striatum; ChP, choroid plexus; CSF, cerebrospinal fluid; CTX, cortex; LV, lateral ventricle; SEZ, subependymal zone; OB, olfactory bulb; EV, extracellular vesicle; CTR, control; ISH, in situ hybridization. Data are shown as mean+/-SEM; each single dot represents an independent biological replicate; significance was tested using Kruskal-Wallis ANOVA; *-p value <0.05, ***-p value <0.001. Scale bars (A) 300 µm, (A') 100µm, (A'') 25 µm, (G) 1 µm, (G', G'') 200 nm.
Choroid Plexus-derived miR-204 Regulates the Number 
of Quiescent Neural Stem Cells in the Adult Brain (2019)
Figure 5

Figure 5. NSCs in the SEZ take up ChP-released miR-204.

(A) Gel pictures of the saturation phase of RT-qPCR analysis for miR-204 in SEZ primary culture cells, ChP co-culture and ChP explants 7 day post preparation.

(B) Gel electrophoresis of RT-qPCR analysis loaded at the saturation phase for miR-204 in SEZ culture medium and SEZ medium from the ChP co-culture.

(C) Dot plot showing the proportion of neural stem cells (GFAP+) out of DAPI+ cells in primary SEZ/ChP co-culture with or without treatment with GW4869 inhibitor or AntimiR204.

(D-G) Micrographs depicting the cellular compositions of SEZ cultures after co-culturing with control treated, GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating. Boxed areas correspond to the higher magnification images in adjacent panels (D', E', F', G').

Data information: See also Fig. EV4. Abbreviations: SEZ, subependymal zone; ChP, Choroid plexus. Data are shown as mean+/-SEM; each single dot represents an independent biological replicate; significance was tested using Kruskal-Wallis ANOVA; *-p value <0.05, ***-p value <0.001. Scale bars (D, E, F, G) 100 µm, (D', E', F', G') 20 µm.
Choroid Plexus-derived miR-204 Regulates the Number 
of Quiescent Neural Stem Cells in the Adult Brain (2019)
Figure 6

Figure 6. ChP specific inhibition of miR-204 decreases neurogenic priming of adult NSCs and the number of qNSCs in the SEZ.

(A) Schematic representation of the experimental setup to address the effect of ChP specific miR-204 inhibition on the number of LRCs and neurogenic priming.

(B, C) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle. (B' and C') are magnifications of the SEZ and (B'' and C'') of the ChP.

(D) Dot plot depicting expression of miR-204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR-204-specific TuD compared to scrambled control.

(E-J) Micrographs depicting immunoreactivity for MEIS2 (E, F) and MCM6 (H, I) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition (F, I) and scrambled control (E, H). White arrows point out the primed LRCs, the arrowheads label the MEIS2 or MCM6+ LRCs. (G, J) Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 (G) and MCM6 (J) after ChP specific miR-204 interference.

(K, L) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control (K) and miR-204 specific TuD (L). Note the reduction of primed LRCs (BrdU+ only, white arrows in K and L) upon TuD-204 injection.

(M) Dot plot depicting the number of LRCs (BrdU+ only) in the SEZ 7 days after miR-204 inhibition.

Data information: See also Fig. EV5. Abbreviations: WT, wildtype; OB, olfactory bulb; CTX, cortex; RMS, rostral migratory stream; SEZ, subependymal zone; dpi, days post injection; ChP, choroid plexus; LV, lateral ventricle; qNSC, quiescent neural stem cell, LRC, label retaining cell; TuD, Tough Decoy. Fluorescent images are full Z-projections of confocal Z-stack, except of K' and L' representing orthogonal projection in the single plane. Data are shown as mean+/-SEM; each single dot represents independent biological replicate; significance was tested using Kruskal-Wallis ANOVA; *-p value <0.05. Scale bars (B, C) 100 µm, (B', B'', C', C'') 50 µm, (E, F, H, I, K, K', L, L') 20 µm.
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 1

Figure 1. PABPC1 and PABPC4 cooperate to support HeLa cell viability.

(A) Western blot analysis wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. siGFP represents a negative control.

(B) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm.

(C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 2

Figure 2. Generation of PABPDHFR HeLa cells.

(A) Schematic diagram of strategy employed to C-terminally tag PABPC1 in PABPC4KO HeLa cells using CRISPR-Cas9 mediated homology directed repair (HDR). Adapted from (Sheridan & Bentley, 2016). Homology arms flanked repair cassettes containing a DHFR sequence, preceded by a HA tag and separated from an antibiotic resistance gene (denoted as AbR and either Puro or Hygro) by a T2A cleavage site followed by a SV40 signal. (B) Western blot confirmation of homozygous PABPC1 tagging with DHFR and degron activity. Successful homozygous tagging of PABPC1 is indicated by the higher molecular weight migration (lane 2) as compared to untagged PABPC1 (lane 1). Degron activity was assessed by culturing PABPDHFR cells in the absence of TMP for 4 or 8 hours and assessing PABPC1 levels by western blotting. (C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm. (D) Western blot analysis of lysates derived from cells imaged in (C).
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 3

Figure 3. Depleting PABP alters transcriptome abundance with a minimal impact on mRNA translation. (A) Ribosome profiles from PABPDHFR cells grown in the presence or absence of TMP for 12 hours. Lysates were subsequently resolved by ultracentrifugation on 5-50% sucrose gradients and fraction were collected during which time UV absorbance at 254nm (Abs 254 nm) was monitored for tracing. 40S and 60S subunits, monosomes (80S) and polysomes positions are indicated above tracings. PABP-expressing and -depleted cells are denoted by a black and blue trace lines, respectively. (B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control). (C) Ribosome profiles from PABPDHFR cells grown in the absence of TMP for 12 hours to deplete PABP and resolved on sucrose gradients containing low KCl [(150 nM) traced in dark blue] or high KCl [(500 mM) traced in turquoise]. (D) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey). (E and F) Top ten Wikipathway (WP) terms (Slenter et al., 2018) significantly enriched among proteins coded for by mRNAs that were decreased (E) or increased (F) in abundance in PABP-depleted cells relative to cells expressing PABP. The number above each column represents the number of genes associated with its corresponding term.
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 4

Figure 4. Characteristics of mRNAs whose abundance is altered following PABP depletion. (A-C) Boxplots of 5´UTR (A), 3´UTR (B) and poly(A) tail lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells).

(D and E) Comparison of log2-fold change in transcript abundances within PABP-depleted cells relative to cells expressing PABP versus their reported P-body log2-fold enrichment status (Hubstenberger et al., 2017). The Pearson's correlation coefficient (R=0.73) and the corresponding p-value (p<2.2e-16) are reported with a corresponding trendline (black) and 95% confidence interval (gray). (D) Points in dark green indicate mRNAs that are downregulated in PABP-depleted cells and depleted from P-bodies. Points in light green indicate mRNAs that are upregulated in PABP-depleted cells and enriched in P-bodies. Transcript numbers in each quadrant are listed. (E) Nuclear-encoded mRNAs encoding cytoplasmic ribosomes (orange), mitochondrial ribosomes (blue) and proteins with mitochondrial functions (green) that are downregulated in PABP-depleted cells relative to PABP-expressing cells and are predominantly depleted from P-bodies. Proteins with mitochondrial functions were defined based on an inventory of human mitochondrial proteins (Calvo et al, 2016).
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 5

Figure 5. Depleting PABP accelerates the decay of stable mRNAs. (A) Boxplots of mRNA half-lives (hrs) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP comparing (Tiana et al., 2020). Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values but not further than 1.5× the inter-quartile range (IQR). (B- H) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 6

Figure 6. Depleting PABP does not impact the decay of a microRNA-targeted reporter mRNA. (A) Schematic representation of the Renilla luciferase (Rluc) reporter mRNAs containing let-7-binding sites (RL-6xB) or mutated seed sites (RL-6xBMUT).

(B) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 7

Figure 7. Depleting mRNA decapping and decay factors suppresses PABP depletion-induced cell death. (A) Western blot analysis PABPDHFR cells depleted of LSM1, DCP2 or XRN1 by siRNA-mediated knockdown. siGFP represents a negative control. (B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.
PABP prevents the untimely decay of select mRNA populations in human cells (2022)
Figure 8

Figure 8. Disrupting the LSM1-7 complex prevents deadenylation-independent decay in PABP-depleted cells. (A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP. (B) PABPDHFR cells were transfected with siRNAs targeting GFP (control) or LSM1. 48 hours later, transfected cells were split and cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. (C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.

EXPANDED VIEW FIGURE LEGENDS
A CRMP4-Dependent Retrograde Axon-to-Soma Death Signal in Amyotrophic Lateral Sclerosis (2021)
Figure 1

Figure 1 -CRMP4 is elevated in ALS diseased spinal cord neurons

(A, B) (A) Representative IHC images and (B) semi quantification of CRMP4 protein in human spinal cords (SC) cross sections from 2 control patients and 3 ALS patients. We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients, Data presented as mean ±SE. DAB: labeled CRMP4. Scale bar: left images 20 µm, right insets 10 µm. Mann-Whitney test ***p = 0.0003.

(C) Representative images of P90 SC cross sections of SOD1G93A and WT mice. Red: denotes NeuN, Green: denotes CRMP4. Scale bar: 10 μm.

(D) Quantification of the percentage of CRMP4 positive SC neurons in

3 WT VS. 3 SOD1G93A mice. We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A, an average of 36 or 41 cells in each repeat respectively. Student's t-test, n = 3, Data presented as mean ±SE, *p = 0.0161.
A CRMP4-Dependent Retrograde Axon-to-Soma Death Signal in Amyotrophic Lateral Sclerosis (2021)
Figure 2

Figure 2 -CRMP4 is mis localized in ALS motor units

(A) Representative images of ALS patient or non-ALS human control intra-muscular nerves. Red: denotes NFH, Green: denotes CRMP4, White: denotes co-localization area using Imaris software. Scale bar: 20 μm.

(B) Quantification of CRMP4 intensity levels in NFH positive intra-muscular distal nerves from 5 non-ALS controls and 4 sALS patients. We analyzed 40 terminal axons from the healthy samples (~8 axons per sample) and 36 terminal axons from sALS samples (~8 axons per sample). Data presented as mean ±SE. Student's t-test, *p = 0.0475.

(C) Representative images of SOD1G93A/ChAT::tdTomato or WTChAT::tdTomato neuromuscular junctions at P90. White: denotes BTX, Red: denotes direct ChAT, Green: denotes CRMP4, Yellow: denotes Z projection of 3D Imaris co-localization of CRMP4 and ChAT. Scale bar: 10 µm.

(D) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice. Total of 44 NMJ's in WT condition and 60 NMJ's in SOD1G93A condition. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0157.

(E) Quantification of the percent of partially denervated NMJ's in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice. We counted 24 NMJ's in SOD1G93A CRMP4 negatives and 67 NMJ's in SOD1G93A CRMP4 positives. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0352.

(F) Representative images of P90 SOD1G93A and WT sciatic nerves. Red: denotes NFH, Blue: denotes GFAP and green denotes CRMP4, Yellow: denotes the Z projection of 3D Imaris co-localization of CRMP4 and NFH. Scale bar: 5 μm.

(G) Quantification of the co-localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis. 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored. Data presented as mean ±SE. Student's t-test, n=3, ****p<0.0001.

(H-I) Western blot analysis and quantification of 3 independent repeats of P90 SN tissues for CRMP4 expression levels (size of ~64 KDa) in SOD1G93A compared to WT. tERK was used as a loading control (size of ~44 KDa). Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0215.