EMBO/soda-nel huggingface dataset:
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 1

Figure 1: Expression of hTauAT causes Tau phosphorylation and mis-sorting in aged mouse hippocampi.

A. The diagram shows the domain structure of wild type human Tau and the location of the substitution A152T (hTauAT). Tau domains are broadly divided into the N- terminal "projection domain" (amino acids M1-Y197) and the C-terminal "assembly domain" (amino acids S198-L441). The C-terminal assembly domain includes three or four pseudo-repeats (~31 residues each, R1–R4), which together with their proline-rich flanking regions (P1 and P2) constitute the microtubule-binding region. Repeat R2 (exon10) and the two near-N-terminal inserts (N1 and N2, exons 2 and 3) may be absent due to alternative splicing.

B. Blots showing expression of hTauAT (hTau, Mr ~67 kDa) and/or endogenous mouse Tau (mTau, Mr ~45-55 kDa) in the hippocampus of 10 month old heterozygous (+/-) and homozygous (+/+) hTauAT mice in comparison to age matched control (Ctrl) detected by pan-Tau antibody K9JA. Actin at 42 kDa serves as loading control.

C. Quantification of (B). Ratio of hTau / mTau indicating a higher hTauAT -expression in homozygous mice (~1.8) compared to heterozygous mice (~1.0). Each bar represents an average of n=4 animals, error bars represent SEM.

D. Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice (n = 4 per group).

E. Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3-region of hetero- and homozygous hTauAT mice at the age of 14 month. Notice the mis-sorted hTauAT in cell bodies and dendrites (arrowheads) of pyramidal neurons of the CA3 region and immunreactivity of the axons (mossy fibers, asterisks). By contrast the control shows no immunoreactivity.

F. Elevated hTauAT -expression causes Tau-pathology inside area CA3 of the hippocampus. Note the increase in Tau phosphorylation probed with the antibody against pT217, a site upstream of the repeat domain. Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3.

G. Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231+pSer235 (arrowheads denote somato-dendritic mislocalized tau).

H. Antibody PHF-1 (phosphorylation sites pSer396+pSer404) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum (asterisks) of area CA3 and pyramidal cell bodies (arrows).

I. Tau phosphorylation detected by antibody AT8 (pS202+pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level.
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 2

Figure 2: Expression of hTauAT leads to pathological conformation and aggregation of Tau in the hippocampus of aged mice.

A. Conformational change of Tau detected by ALZ-50 antibody immunoreactivity in stratum lucidum (asterisks) of area CA3 and somata in stratum pyramidale (arrowheads).

B. Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero- and homozygous hTauAT mice at 14 month of age compared to non-reactive control littermate mice. In homozygous hTauAT mice the extent of neurofibrillary tangles (NFTs) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice (white arrowheads). The control shows no silver-reactive Tau aggregates.

C. Western blot analysis using the pan-Tau antibody K9JA indicates sarcosyl-insoluble Tau species of human hTauAT (upper band) and mouse Tau (mTau, lower band). Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice.

D. Quantification of (C). The ratio of hTauAT /mTau indicates a stronger aggregation in the homozygous hTauAT mice (~1.4) compared to heterozygous hTauAT mice (~0.7 (n=4 animals, error bars represent SEM).

E. Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density (asterisks) in a control animal at 12 months. (ds) dendritic spine, (pt) presynaptic terminal and (m) mitochondrium.

F. Example of a degenerating synaptic bouton (arrow) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density (asterisks) in a transgenic animal at 13 months.

G. Electron dense neuronal cytoplasm with dark nucleoplasm and various vacuoles (arrow) detected in CA3 pyramidal neurons (arrow) at 13 months in transgenic animals in proximity to normally appearing neurons (asterisks), degenerating apical dendrite (d).

H. Neuropil of a transgenic animal (13 months) with a degenerating neuritic profile (arrow).

I. Degenerating dendrite (arrow) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria (arrow).

J. Degenerating dendrite containing electron-dense whorling membrane fragments (arrow) in proximity to normally appearing synapses (synapse with a perforated postsynaptic density, asterisks).
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 3

Figure 3: hTauAT expression causes Tau phosphorylation and mis-sorting in organotypic hippocampal slice cultures.

A. Western blot analysis with pan-Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau (mTau). Slice homogenates from non-transgenic littermate (lane 1, Ctrl), heterozygouse hTauAT (lane 2, (+/-)) or homozygouse hTauAT slices (lane 3, (+/+)) were analyzed after three weeks in culture. Actin served as a loading control.

B. Quantification of hTauAT expression levels (n=3-6 slice homogenates). The ratio between hTauAT and endogenous mouse Tau is shown.

C. Immunohistochemistry with antibodies against human Tau (TauY9 antibodies; middle panel) and dendritic marker MAP2 (left panel) in slice cultures at DIV 10. Stratum radiatum of area CA3 is shown. Although MAP2 staining is apparent, no signal is detected by the TauY9 antibody in control slices. In contrast, in hTauAT slices human Tau is detected in both dendrites and axons (see asterisk in merged image).

D. Immunohistochemistry against phosphorylated Tau at the PHF1-epitope in area CA3 of a control (upper lane) or hTauAT slice (bottom lane). PHF1 staining is slightly visible in some axons in control slices. In contrast, PHF1-phosphorylated Tau is apparent in somata, dendrites and axons of neurons in hTauAT slice cultures.

E. Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 (red) and Tau (K9JA antibody, green). Tau staining is seen in dendritic spines (asterisks) in contrast to MAP2, which is restricted to the dendritic shaft.
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 4

Figure 4: hTauAT causes elevation of Ca++ levels in CA3 hippocampal neurons at resting state and after membrane depolarization through extrasynaptic NMDA receptors.

A. Slices (DIV15) from control mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca++ ([Ca++]i) after KCl application.

B. Slices (DIV15) from hTauAT mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images at baseline and after application of high potassium in area CA3 are depicted. Note the increase in [Ca++]i (false color legend) under both conditions in hTauAT slices compared to controls.

C. Electrophysiological example traces (control = black; hTauAT = red) of excitatory postsynaptic field potentials (fEPSP) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca++]i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2-AM loaded hippocampal slices. After depolarization [Ca++]i rises up to ~600 nM in hTauAT slices (red trace, n=6 slices, prepared from at least three animals), compared to ~300 nM in control slices (black trace, n=6 slices; prepared from at least three animals). Note that even under resting conditions [Ca++]i is elevated to ~120nM due to hTauAT expression.

D. Quantification of the maximum [Ca++]i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions [Ca++]i was elevated in transgenic slice cultures (~120 nM, n=11) compared to controls (~80 nM, n=8). Slices were incubated with one of the following channel blockers: Nifedipine (NIF, VGCC; Ctrl n=8; A152T: n=7), APV (NMDAR; Ctrl n=7; A152T n=8), ifenprodil (IFEN, NMDAR; Ctrl n=8; A152T n=7), memantine (MEM, NMDAR; Ctrl/A152T n=6), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPAR; Ctrl n=8; A152T n=7)), tetrodotoxin (TTX, VGNaC; Ctrl n=4; A152T n=5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n=6) or in Ca++ free buffer (Ctrl n=10; A152T n=7). For each experiment slice preparations from at least three different mice were used. One-way ANOVA followed by Tukey’s post-hoc test; Ctrl: F(7/51) .3,533; p=0.0036; A152T: F(8/55)=5,230; p<0.0001.

For an overview of drug concentrations see EV Table 1.

E. Quantification of the maximum peak increase in intracellular calcium in the same slices than in (D) after high KCl stimulation. One way ANOVA followed by Tukey’s post-hoc test Ctrl: F(7/45)=4,080; p=0.0015; A152T: F(8/53)=7,580; p<0.0001).

Error bars represent SEM; One-Way ANOVA *p<0.05; **p<0.01 and ***p<0.001.
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 5

Figure 5: hTauAT expression enhances extracellular glutamate, cytotoxicity and progressive neuronal loss in the hippocampus.

A. Glutamate content in the culture medium ([Glu]e) from either hTauAT (n=10 culture inserts containing 6 slices each) or control slices (n=5-12 culture inserts) analyzed at DIV 5, 10, 20 and 25. Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point. Increased levels were observed between DIV 5 and DIV 20. At DIV 25 glutamate levels already declined to control levels. One-way ANOVA followed by Tukey’s post-hoc test *p<0.05; **p<0.01 ***p<0.001.

B. Lactate dehydrogenase (LDH) release (a measure of cytotoxicity) analyzed in hTauAT (n=17-19 culture inserts containing 6 slices each) or control slices (n=15-16 culture inserts) at DIV 5, 10, 20 and 25. LDH release of hTauAT slices is shown in percent of control at each time point. A pronounced increase (~25%) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One-way ANOVA: *p<0.05; **p<0.01 and ***p<0.001.

C. Slice cultures from control (left), hTauAT (middle) or hTauAT treated with ceftriaxone (CEF; 100 µM; right) stained against neuronal nuclear protein (NeuN) at DIV 30. Representative neuronal cell layers are shown. hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 (middle panel, red arrows).Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3.

D. NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest (ROI). The number of neurons was reduced in hTauAT slices, both within area DG and CA3, whereas the number of neurons in the CA1 was only slightly affected. Neuronal loss was largely prevented in cultures treated with ceftriaxone (CEF) in dentate gyrus and area CA3 (n=14-19 slices per group and area; prepared from at least 6 animals). One-way ANOVA followed by Tukey’s post-hoc test *p<0.05; **p<0.01
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 6

Figure 6: hTauAT enhances synaptic transmission and dendritic spine density in area CA3 but does not interfere with synaptic plasticity.

A. Representative microphotograph of spines on dendrites from control littermate mice at the age of 12 months. Apical dendrites from CA3 pyramidal neurons are visualized by Golgi staining. The contrast of photomicrographs was inverted.

B. Dendritic spines from heterozygous hTauAT mice, seen by Golgi staining.

C. Quantification of dendritic spine densities in control littermate and hTauAT mice. Note that the spine density in heterozygous hTauAT is increased (control littermates: n = 2 animals; heterozygous hTauAT n = 4 animals; an average of 5 dendrites of CA3 pyramidal neurons were analyzed per animal; unpaired t-test).

D. Input / output curve of field potential recording in the mossy fiber pathway in acute slices of 12±1 month old mice demonstrating significantly enhanced basal synaptic transmission (Multiple repeat ANOVA,F(9, 126)=2.00, p=0.044) in area CA3 in mice expressing hTauAT. Scale bars: 500 µV vertical bar and 20 msec horizontal bar.

E. Long-term potentiation in the mossy fiber pathway demonstrates no differences in long-term plasticity between control (black) and hTauAT expressing mice (red).

F. Input / output curve of field potential recording in stratum pyramidale (s.p.) of area CA3 in organotypic hippocampal slices at DIV 30. Somatic field potentials are increased in hTauAT compared to control littermates. Scale bars: 800 µV vertical bar and 20 msec horizontal bar.

Error bars represent SEM; t-test: *p<0.05; **p<0.01 and ***p<0.001.
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 7

Figure 7: Cultured slices expressing hTauAT show a progressive increase in epileptiform activity and mossy fiber sprouting which can be prevented by CEF application.

A. Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice (DIV30) during wash-in of 50 µm picrotoxin. This leads to blockage of GABAergic transmission followed by epileptiform discharges (burst) occurring approximately after 3 min. The zoom-in reveals that such an epileptiform discharge (burst) consists of repetitive firings.

B. Similar example trace as in (A) recorded in a hTauAT expressing slice (red) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression.

C. Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~2-fold increase in hTauAT slices (n=8 slices per group).

D. Firings per burst are increased ~1.5-fold already at the early time point DIV10 due to hTauAT expression.

E. Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10. Burst frequency is enhanced in hTauAT expressing slices (n = 10 slices). If Ceftriaxone (CEF) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented (n = 5 slices).

F. Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30. Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation (n = 6 slices).

G. Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN (red) and pan-Tau (K9JA antibody, green). Axonal projections (green) of granule cells (red) in the dentate gyrus are depicted.

H. Hippocampal slice culture expressing hTauAT stained against NeuN (red) and pan-Tau (green) showing mossy fiber sprouting in the hilar region, CA3 and dentate gyrus (white arrows).

Error bars represent SEM; t-test: *p<0.05; **p<0.01 and ***p<0.001.
The Tau/A152T mutation, a risk factor for frontotemporal spectrum disorders, leads to NR2B receptor-mediated excitotoxicity (2016)
Figure 8

Figure 8: The pathophysiological phenotype of hTauAT expression is related to enhanced presynaptic release and is prevented by increasing EAAT2/GLT1 activity.

A. Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin (TeNT) or 1 µM tetrodotoxin (TTX) at DIV5. Under both conditions, glutamate levels in the culture medium of hTauAT slices decreased to control levels (n=6 culture inserts containing 6 slices each / group and condition).

B. Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV1-DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices (n=8-9 culture inserts containing 6 slices each / group and condition). One-Way ANOVA F(3,30)=14,89; p<0.001

C. Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV 1 to DIV 20 (n=6-12 culture inserts containing 6 slices each / group and condition). One-Way ANOVA followed by Tukey’s post-hoc test.

D. Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated (n=7-8 culture inserts containing 6 slices each / group and condition) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices. One-Way ANOVA F(3,25)=9.075; p=0.0003).

E. LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures (n=8-10 culture inserts containing 6 slices each / group and condition). One Way ANOVA F(3,32)=7.685; p=0.0005).

F. Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days (n=5-9 slices; prepared from at least three animals). One way ANOVA F((3,25)=7.317; p=0.0011).

Error bars represent SEM; One-Way ANOVA followed by Tukey’s post-hoc test *p<0.05; **p<0.01 and ***p<0.001.
CDKL5 kinase controls transcription-coupled responses to DNA damage (2021)
Figure 1

Figure 1. CDKL5 senses DNA damage in a PAR dependent manner.

A. BrdU-sensitized U-2-OS Flp-In T-REx cells stably expressing mCherry-FAN1 and GFP-NLS or GFP-CDKL5 (no NLS) were line micro-irradiated (355 nm) and imaged after 2 min. Scale bar is 10 μm

B. BrdU-sensitized U-2-OS cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273 (0.3 μM) for 1 h prior to micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μm

C. Same as B. except that cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM) or PDD00017273 (0.3 μM) for 1 h prior to spot micro-irradiation (405 nm). Individual cells from one of two independent biological replicates is shown. Scale bar is 10 μm

D. Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point.

E. BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ, PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μm

F. Diagram of the workflow for the chromatin retention experiments.

G. Cells subjected to the workflow in F. were detergent-extracted and fixed before staining with anti-GFP or fibrillarin (nucleoli). Scale bar is 10 μm

H. Quantification of the detergent-insoluble GFP-NLS-CDKL5 signal (minus nucleolar signal). The mean ± SD from three biological experiments is shown. Statistical significance was assessed by one-way-ANOVA-test. Asterisks ** indicate P-value of <0.01; ns - not significant.
CDKL5 kinase controls transcription-coupled responses to DNA damage (2021)
Figure 2

Figure 2. The CDKL5 recruitment domain binds PAR directly.

A. Schematic diagram of CDKL5 deletion mutants, deleting from the N-terminal (blue) or C-terminal (black) ends. All proteins were expressed with an N-terminal NLS and GFP tag.

B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL) GFP-NLS-CDKL5 were subjected to line micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μm

C. Schematic for fragments corresponding to the PAR dependent recruitment region in CDKL5 as identified in B.

D. Same as in B except that the GFP-NLS tagged CDKL5 fragments indicated were examined. Scale bar is 10 μm

E. Coomassie gel showing recombinant fragments of human CDKL5 fused to GST purified from bacterial lysates. GST and APLF were also purified as controls.

F. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on nitrocellulose membrane and then incubated with synthetic PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown

(G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting using the indicated antibodies. One of two independent experiments is shown
CDKL5 kinase controls transcription-coupled responses to DNA damage (2021)
Figure 3

Figure 3. CDKL5 recruitment to DNA lesions requires ongoing transcription.

A. (Top) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. The "mock" trace shown is identical to the "mock" trace shown in Fig. 1D, as the data come from the same set of biological replicates. Scale bar is 10 μm

(B-D) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μm

E. Same as B-D. except that BrdU-sensitized cells stably expressing GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μm

F. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm