Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , Schematic representation of isolating primary astrocyte and primary neuron cultures from postnatal day 1 (P1) rat cortex. b , qPCR data showing significantly higher expression of rAtp13a4 in rat primary cortical astrocytes compared to neurons. n = 3 independent cultures. Paired t test. c , Detection of msAtp13a4 in cortical astrocytes by RNA FISH in Aldh1L1-EGFP mice at P14. d , Quantification of total and astrocyte-specific msAtp13a4 expression across all cortical layers (L1, L2/3, L4, and L5) of the V1 cortex at P7, P14, and P21. Astrocytic msAtp13a4 mRNA expression was quantified by normalizing the volume of msAtp13a4 mRNA puncta with the astrocyte soma (labeled by eGFP). 3 sections per animal, 3 animals per time point. Scale bar, 10 μm. e , Astrocyte-specific AAVs (serotype PHP.eB) were used to express Lamp1 or nonspecific cytosolic control. NES, nuclear export sequence; ITR, inverted terminal repeats; GfaABC1D truncated GFAP promoter; HA, hemagglutinin tag; Turbo, TurboID biotin ligase; pA, polyadenylation. f , Outline of the experimental paradigm. n = 3 biological replicates per construct per genotype (1 replicate = 2 animals per pooled sample). g , Schematic of Lamp1 protein localization within astrocyte perisynaptic process (PAP). h , Volcano plot showing the differential abundance of proteins detected by Astro-Lamp1-TurboID compared to Astro-Cyto-TurboID. Dotted lines indicate threshold values FC > 1.5 (log2FC > 0.58), p-value < 0.05 (log10p < −1.3). i , Empirical cumulative distribution function (eCDF) listing potential endo-lysosomal polyamine transporters (red dots) among the most abundant proteins detected by Astro-Lamp1-TurboID. j , Schematic of the catalytic cycle of the archetypical P5B ATPase ATP13A2. k , Dose-response curves showing the effect of putrescine (PUT), spermidine (SPD) and spermine (SPM) on the ATPase activity of solubilized purified ATP13A4 (WT or catalytically dead p.D486N mutant). The number of independent biological experiments was as follows: n = 3 (D486N PUT, SPD, SPM), n = 5 (WT PUT), n =7 (WT SPD) and n = 4 (WT SPM). l-m , ATP13A4 phosphoenzyme (EP) levels in control (CON) conditions and in the presence of PUT, SPD, SPM, or hydroxylamine (HA). l , Representative autoradiogram. m , Quantification of EP. n = 5 independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test. n , Schematic illustrating the BODIPY-polyamine uptake assay in primary astrocytes with ATP13A4 knockdown. o , shRNA targeting mouse/rat Atp13a4 efficiently reduces Atp13a4 expression in astrocytes compared to control in which the shRNA sequence was scrambled (shCtrl). n = 3 independent cultures. Paired t test. p-q , Confocal microscopy of BODIPY-labelled SPD distribution. Scale bar, 10 μm. Mean fluorescence intensities (MFI) of BODIPY-SPD (shCtrl, 20 cells; shAtp13a4, 18 cells). n = 3 independent cultures. Paired t test.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: Expressing, Labeling, Control, Sequencing, Construct, Activity Assay, Purification, Mutagenesis, Knockdown, shRNA, Confocal Microscopy, Fluorescence

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , Representative images of in vivo expression in the cortex of different TurboID constructs labeled with HA and the biotinylating activity labeled with streptavidin. Merged images show the colocalization of HA and biotin signals in astrocytes. Scale bar, 1000 μm. b - c , HA immunoblotting verifies expression of Astro-Cyto-TurboID ( b ) and Astro-Lamp1-TurboID ( b’ ). Streptavidin blotting verifies biotinylation activity of Astro-Cyto-TurboID ( c ) and Astro-Lamp1-TurboID ( c’ ) in cortical lysates (input) and subsequent immunoprecipitation (binding). d , Principal component analysis (PCA) of BioID data shows Astro-Lamp1-TurboID samples (purple) clustering separately from Astro-Cyto-TurboD samples (gray) and study pool QC (SPQC) samples (green). e , Heatmap displaying the correlation of protein abundance for all pairwise combinations of samples. Unsupervised hierarchical clustering shows Astro-Lamp1-TurboID samples clustering separately from Astro-Cyto-TurboID and SPQC samples. f , Bar graph depicting the detection ratio of Lamp1-TurboID versus Astro-Cyto-TurboID for putative polyamine transporters (Atp13a3 and Atp13a4) and selected endo-lysosomal proteins. ( g - h ) Bars show the top 10 most significant Gene Ontology (GO) terms, ordered by lowest adjusted p-value, for the proteins differentially detected by Astro-Lamp1-TurboID compared to Astro-Cyto-TurboID. ( g ) Cellular Compartment; ( h ) Biological Process.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: In Vivo, Expressing, Construct, Labeling, Activity Assay, Western Blot, Immunoprecipitation, Binding Assay, Quantitative Proteomics

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , Structural visualization of hATP13A4 (AlphaFold prediction) and hATP13A2 (PDB 7n78) using PyMOL. Top: overlay of hATP13A4 (blue) and hATP13A2 (green), with spermine shown in grey. Bottom: close-up view of the spermine binding pocket. b - g , Coomassie staining ( b , d , f ) and Western blot analysis ( c , e , g ) showing the purification process for WT hATP13A4 ( b - c , f - g ) or the p.D486N mutant ( d - e ), starting from detergent-solubilized HEK293T cell lysates ( b - e ) or yeast membranes ( f - g ), followed by streptavidin affinity chromatography and subsequent elution with biotin. n ≥ 3 independent purifications. FT, flow-through; W, wash; E, elution. h , Representative autoradiogram showing phosphoenzyme (EP) formation of WT hATP13A4 in yeast membrane fractions (heavy membrane fraction P2 and light membrane fraction P3) and purified protein, which is sensitive to 0.3 M hydroxylamine (HA). i - j , Representative autoradiogram ( i ) and quantification ( j ) of pulse ([γ- 32 P]-ATP) chase (cold ATP or ADP) experiment to determine ATP and ADP sensitivity of hATP13A4 EP. n = 3 independent experiments. k - l , Representative autoradiogram ( k ) and quantification ( l ) of pulse ([γ- 32 P]-ATP) chase (cold ATP) experiment in the presence and absence of spermidine (SPD) or spermine (SPM) in yeast membranes. n = 4 (ATP + SPM) or 5 (ATP, ATP + SPM) independent experiments. m - n , Representative autoradiogram ( m ) and quantification ( n ) of purified ATP13A4 EP levels in the presence of 0.3 M HA, 1 mM SPD or SPM. n = 4 independent experiments. One-way ANOVA with Dunnett’s post-test.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: Binding Assay, Staining, Western Blot, Purification, Mutagenesis, Affinity Chromatography, Membrane

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , Schematic of astrocyte-neuron co-culture used to assess astrocyte morphology. b , Representative images of astrocytes (green) transfected with either shRNA targeting mouse/rat Atp13a4 (shAtp13a4) or a scrambled control (shCtrl), co-cultured with WT neurons (not visible) for 48 h. Scale bar, 50 μm. c , Quantification of astrocyte complexity via Sholl analysis for conditions in b . n = 72 astrocytes per condition from three independent experiments. d , Representative images of shRNA expressing astrocytes (green) in the presence or absence of an shRNA-resistant mouse Atp13a4 (msAtp13a4) construct with an N-terminal mCherry tag (red) after 48 h co-culture with WT neurons. Scale bar, 50 μm. e , Quantification of astrocyte complexity for conditions in d . n = 60 astrocytes per condition from three independent experiments. f , Representative images of shRNA expressing astrocytes co-cultured with methanol-fixed WT neurons for 48 h. Scale bar, 50 μm. g , Quantification of astrocyte complexity for conditions in f . n = 60 astrocytes per condition from three independent experiments. c , e , g , Mixed effects ANOVA ( c , g ) with Tukey’s post-hoc test ( e ). h , Schematic of PALE (Postnatal Astrocyte Labeling by Electroporation). Plasmids were injected into the lateral ventricle of CD1 mice at late P0, followed by electroporation into radial glial stem cells, leading to sparse knockdown and labeling of cortical astrocytes. i , Representative images of in vivo shRNA-transfected astrocytes (labelled with mCherry) at P21. j , Quantification of in vivo astrocyte territory volumes by convex hull analysis in Imaris. Only astrocytes from V1 cortex were imaged and analyzed. The average astrocyte territory volume of individual mice is plotted in black (shCtrl) or red (shAtp13a4). n = 18–22 astrocytes from 3-4 mice, nested t test. All data are presented as mean ± SEM, except in j where individual data points are shown.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: Co-Culture Assay, Transfection, shRNA, Control, Cell Culture, Expressing, Construct, Labeling, Electroporation, Injection, Knockdown, In Vivo

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , shRNA-resistant ATP13A4 construct with silent mutations (red) in the shRNA target site to prevent knockdown. b , Representative images of astrocytes (green) transfected with shRNA targeting mouse/rat Atp13a4 (shAtp13a4) or a scrambled control (shCtrl), co-cultured with WT neurons (not labeled) for 48 h, and treated with spermidine during the final 8 h of co-culture. Scale bar, 50 μm. c , Quantification of astrocyte complexity via Sholl analysis for conditions in b . n = 46-68 astrocytes per condition from three independent experiments. d , Representative images of shRNA expressing astrocytes co-cultured with WT neurons for 48 h and treated with spermine during the final 8 h of co-culture. Scale bar, 50 μm. e , Quantification of astrocyte complexity for conditions in d . n = 50-60 astrocytes per condition from three independent experiments. f , Representative images of shRNA expressing astrocytes (green), treated with spermidine for 24 h and then co-cultured with WT neurons for 48 h. Scale bar, 50 μm. g , Quantification of astrocyte complexity for conditions in f . n = 60-63 astrocytes per condition from three independent experiments. h , Representative images of shRNA expressing astrocytes (green), treated with spermine for 24 h and then co-cultured with WT neurons for 48 h. Scale bar, 50 μm. i , Quantification of astrocyte complexity for conditions in h . n = 60 astrocytes per condition from three independent experiments. All data are presented as mean ± SEM. c , e , g , i , Mixed effects ANOVA with Tukey’s post-hoc test.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: shRNA, Construct, Knockdown, Transfection, Control, Cell Culture, Labeling, Co-Culture Assay, Expressing

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , Schematic of neuronal culture assay to evaluate the effects of astrocyte-conditioned medium (ACM) on synapse formation. b , Excitatory synapses marked with Bassoon and Homer1. Scale bars, 20 μm. c , Quantification of excitatory synapses (Bassoon and Homer1 co-localization) in b . n = 58-64 cells per condition from three independent experiments. Individual data points are shown, with the average of each experiment plotted in grey (no ACM), black (shCtrl ACM) or red (shAtp13a4 ACM). Nested one-way ANOVA with Tukey’s post-test. d , Metabolomics analysis of spermidine levels in shCtrl ACM versus shAtp13a4 ACM. n = 3 independent experiments. Unpaired two-tailed t test. e , Excitatory synapses marked with Bassoon and Homer1 in neuronal cultures treated with spermidine (SPD) or spermine (SPM). Scale bars, 20 μm. f , Quantification of excitatory synapses (Bassoon and Homer1 co-localization) in e . n = 64-66 cells per condition from three independent experiments. c , f , Individual data points are shown, with the average of each experiment plotted in grey (no ACM), black (shCtrl ACM), red (shAtp13a4 ACM), green (SPD) or blue (SPM). Nested one-way ANOVA with Tukey’s multiple comparisons test. g , Schematic of Atp13a4 KO mice, illustrating the effects at the protein level. h , Genomic PCR from Atp13a4 WT, heterozygous and KO mice. i , Excitatory synapses (Bassoon and Homer1 co-localization) in V1 cortex of Atp13a4 WT and KO mice. White arrows indicate excitatory synapses. Scale bar, 10 μm. j , Quantification of excitatory synapse numbers in Atp13a4 WT and KO mice. n = 8 (KO), 10 (WT) mice with 2 sections/mouse and 3-4 images/section. Data points represent mouse averages. Unpaired two-tailed t test. k , Representative mEPSC (miniature excitatory postsynaptic currents) traces from V1 cortex in acute brain slices of WT and Atp13a4 KO mice. l , Quantification of frequency average (inset) and cumulative probability of mEPSC from Atp13a4 WT and KO neurons. m , Quantification of amplitude average (inset) and cumulative probability of mEPSC from Atp13a4 WT and KO neurons. l - m, n = 26 (WT), 23 ( Atp13a4 KO) neurons from 3 mice per genotype. Mann-Whitney U test (inset). Kolmogorov-Smirnov test (cumulative probability). Individual data points are shown.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , Representative images of neurons transfected with GFP and treated with astrocyte-conditioned medium (ACM). Scale bar: 100 μm. b , Quantification of dendrite tree length in a . n = 52-60 cells per condition from three independent experiments. Nested one-way ANOVA with Tukey’s post-test. c - d , Quantification of Bassoon ( c ) and Homer1 ( d ) puncta of neuronal cultures treated with ACM , n = 58-64 cells per condition from three independent experiments. Nested one-way ANOVA with Tukey’s multiple comparisons test. e - g , Metabolomics analysis of ornithine ( e ), putrescine ( f ) and spermine ( g ) levels in shCtrl ACM versus shAtp13a4 ACM. n = 3 independent experiments. Unpaired two-tailed t test. h-i , Quantification of Bassoon ( h ) and Homer1 ( i ) puncta of neuronal cultures treated with SPD or SPM . n = 64-66 cells per condition from three independent experiments. b - d , h - i , Individual data points are shown, with the average of each experiment plotted in grey (no ACM), black (shCtrl ACM), red (shAtp13a4 ACM), green (SPD) or blue (SPM). Nested one-way ANOVA with Tukey’s multiple comparisons test. j , Representative mIPSC (miniature inhibitory postsynaptic currents) traces from V1 cortex in acute brain slices of WT and Atp13a4 KO mice. k , Quantification of frequency average (inset) and cumulative probability of mIPSC from Atp13a4 WT and KO neurons. l , Quantification of amplitude average (inset) and cumulative probability of mIPSC from Atp13a4 WT and KO neurons. k - l, n = 26 (WT), 23 ( Atp13a4 KO) neurons from 3 mice per genotype. Mann-Whitney U test (inset). Kolmogorov-Smirnov test (cumulative probability). Individual data points are shown.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: Transfection, Two Tailed Test, MANN-WHITNEY

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a , Sequence alignment showing disease-associated residues are highly conserved. The alignment was generated using Clustal Omega . b , Predicted 3D structure of ATP13A4 (AlphaFold) with disease-associated residues indicated. c-f , Coomassie staining ( c , e ) and Western blot analysis ( d, f ) showing the purification process for the disease-associated ATP13A4 variants p.A356V ( c-d ) and p.E646D ( e-f ), starting from detergent-solubilized HEK293T cell lysates, followed by streptavidin affinity chromatography and subsequent elution with biotin. n ≥ 3 independent purifications. FT, flow-through; W, wash; E, elution. g - h , Phosphoenzyme (EP) levels of purified, solubilized ATP13A4 (WT and disease-associated variants p.A356V and p.E646D) under control (CON) conditions and in the presence of putrescine (PUT), spermidine (SPD), spermine (SPM), or hydroxylamine (HA). g , Representative autoradiogram. h , Quantification of EP in g . n = 5 independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test. Individual data points are shown. i , Human RNA-seq data (from ) showing that ATP13A4 is differentially downregulated in human epilepsy astrocytes [astrocyte temporal lobe epilepsy (TLE) versus astrocyte temporal lobe (TL) control].

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: Sequencing, Generated, Staining, Western Blot, Purification, Affinity Chromatography, Control, RNA Sequencing

Journal: medRxiv

Article Title: Astrocytic polyamine transport by ATP13A4 tunes excitatory synaptic transmission

doi: 10.1101/2025.04.04.25325117

Figure Lengend Snippet: a-c , Dose-response curve showing the effect of putrescine ( a ), spermidine ( b ) and spermine ( c ) on the ATPase activity of solubilized purified ATP13A4 (WT or disease-associated p.A356V and p.E646D variants). n = 4 independent experiments. d - g , The open field test. d , Schematic of the open field test arena, showing the center and periphery zones. e , Time spent in the center of the arena for each group. f , Center time analyzed in 30 s time bins for females. A global nonlinear regression tested whether a single curve fit all datasets (null) or if separate curves were required (alternative). g , Time spent in the periphery (thigmotaxis) for each group. h - j , The Morris water maze test. h , Schematic representation of the Morris water maze (MWM) with 10 training days and a probe trial on day 6 and day 11. T, the target quadrant where the hidden platform is normally located; A1 and A2, the adjacent quadrants that lie on either side of the target quadrant; and OQ, the opposite quadrant, which is diagonally across from the target. MWM acquisition curves (distance to the hidden platform) in female ( i ) and male ( j ) cohorts, respectively, over 10 days of training. k - m , The rotarod test. k , Schematic of the rotarod apparatus for motor coordination testing. Latency to fall from the rotating drum over four test trials in female ( l ) and male ( m ) mice; percentages at the bottom indicate the proportion of mice that reached the maximum latency. n - q , Immobility time following PTZ (pentylenetetrazol) challenge in female ( n , p ) and male ( o , q ) mice. Immobility duration is analyzed in 60 s time bins ( n - o ) and longest immobility episode is analyzed in 3 min time bins ( p - q ). r , Illustration of the role of ATP13A4 in astrocytic spermidine uptake and its impact on astrocyte arborization and synaptogenesis. Data are presented as mean with individual data points shown ( e , g , l , m ) or mean ± SEM. Statistical comparisons were made using unpaired t tests ( e , g ), mixed-effects analysis (REML) with Dunnett’s multiple comparisons test ( i , j , n , o ), two-way RM ANOVA with Šidák’s post-test ( p - q ) and Mann-Whitney U test ( l , m ). Schematic in panel r was created with bioRender.com.

Article Snippet: Custom-made probes for Atp13a4 were acquired from Molecular Instruments.

Techniques: Activity Assay, Purification, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Screening of homing and tissue-penetrating peptides by microdialysis and in vivo phage display

doi: 10.26508/lsa.202201490

Figure Lengend Snippet: Reagents and tools table.

Article Snippet: The push syringe was connected to the inlet spike of the CMA 12 custom-made 3-MDa probe or CMA 12 1-MDa probe (both from CMA Microdialysis AB) and the outlet spike to the pull syringe via CMA FEP tubing (CMA Microdialysis AB).

Techniques: Recombinant, Sequencing, Next-Generation Sequencing, Staining, Plasmid Preparation, Software, Microscopy