Journal: bioRxiv

Article Title: Targeting the ClpP-αSynuclein Interaction with a Decoy Peptide to Mitigate Neuropathology in Parkinson’s Disease Models

doi: 10.1101/2025.02.16.638540

Figure Lengend Snippet: Primary cortical neurons were isolated from E18 mouse cortex (see methods) and plated on coverslips (Day 0). Neurons were treated with human α-Syn PFF on Day 5 at 50ng/ml. On Day 7, TAT or CS2 (1µM) was added daily till Day 12. (A) Neurons were fixed in 4% PFA and subjected for immunostaining with anti-ClpP, anti-α-Syn, and anti-NeuN. Shown are the representative Z-stack images of 3 independent experiments. Colocalization of ClpP with α-Syn were examined. N=12 neurons. (B) Histogram: relative Mandel’s co-efficiency and pearson’s co-efficiency for ClpP and α-Syn colocalization. (C) Neurons were fixed in 4% PFA and subjected for immunostaining with anti-Tuj1 and anti-pS129. Shown are the representative images of at least 3 independent experiments. (D) Histogram: related fluorescence intensity of pS129-α-Syn per Tuj1+ area. (E) Neurons were fixed in 4% PFA/4% sucrose/1%Triton-X100 to extract the soluble factions, and subjected for immunostaining with anti-ClpP, anti-α-Syn, and anti-NeuN. Shown are representative Z-stack images of 3 independent experiments. (F) Histogram: relative fluorescence intensity of ClpP in the neuronal insoluble fractions. At least 30 neurons were counted. (G) Neurons were fixed in 4% PFA and subjected for immunostaining with anti-pS129, anti-Synapsin1, and anti-PSD95 antibodies. Shown are the representative images of 3 independent experiments. (H) Histogram: Pearson’s co-efficiency of PSD95/Synapsin1 colocalization. At least 30 areas were counted. Scale bar=30µm. All data are expressed as the mean ± SEM. Data are compared with one-way ANOVA with Tukey’s post-hoc test in D, F, H. Unpaired student t -test is used in B.

Article Snippet: Antibody against Synapsin1 (106104, 1:500) was from SYSY Synaptic System.

Techniques: Isolation, Immunostaining, Fluorescence

Journal: bioRxiv

Article Title: Targeting the ClpP-αSynuclein Interaction with a Decoy Peptide to Mitigate Neuropathology in Parkinson’s Disease Models

doi: 10.1101/2025.02.16.638540

Figure Lengend Snippet: A53T α-Syn patient iPSCs (A53T) and its isogenic control iPSCs (ISO) were differentiated into DA neurons (see methods) for 28 days. Neurons were treated with TAT or CS2 for 5 consecutive days starting from Day 28 at 1µM. (A) Neurons were fixed and subjected for immunostaining with anti-TH, anti-pS129 antibodies. Shown are the representative images of at least 5 independent experiments. Enlarged area are circled in white. (B) Histogram: related pS129-α-Syn intensity per TH+ area. (C) Neurons were fixed and subjected for immunostaining with anti-PSD95 and anti-Synapsin1 antibodies. Shown are the representative images of 6 independent experiments. Enlarged neuronal dendrites were circled in yellow. (D) Histogram: relative fluorescence intensity of PSD95 per MAP2 length. (E) Histogram: relative fluorescence intensity of Synapsin1 per MAP2 length. Scale bar=30µm. All data are expressed as the mean ± SEM. Data are compared with one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: Antibody against Synapsin1 (106104, 1:500) was from SYSY Synaptic System.

Techniques: Control, Immunostaining, Fluorescence

Journal: Acta Neuropathologica Communications

Article Title: Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils

doi: 10.1186/s40478-023-01575-0

Figure Lengend Snippet: Uptake of α-synuclein fibrils following PFFs intravitreal injection. A TEM images of mouse PFFs. Scale bar: 500 nm. B Thioflavin-T intensity of fibrils at day 7 (n = 2 measurements). C CD spectra of α-synuclein monomers and PFFs. D Immunohistochemical staining of total α-synuclein in the retina of PFF-injected mice 1, 3, and 7 days after injection. E – G Quantification of immunoreactivity to α-synuclein in the IPL at 1 day ( E ), 3-days ( F ), and 7-days ( G ) after injection. Scale bar: 50 µm. Data in E , F , G , I and J are expressed as means ± s.e.m. (PBS, n = 5 mice; PFF, n = 5 mice; * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test

Article Snippet: Later, samples were incubated overnight at room temperature with primary antibodies against α-synuclein (Syn1, 1:1000; BD Transduction, 610,787 clone 42/ α-synuclein, BD Biosciences, Franklin Lakes, NJ, USA), pS129 synuclein (1:500; Abcam, EP1536Y clone, ab51253), AP2 α (Invitrogen, 3B5 MA1-872, Waltham, MA, USA) in 5% goat serum.

Techniques: Injection, Immunohistochemical staining, Staining

Journal: Acta Neuropathologica Communications

Article Title: Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils

doi: 10.1186/s40478-023-01575-0

Figure Lengend Snippet: Intravitreal injection of α-synuclein fibrils leads to pS129 accumulation in the retina. A pS129-positive inclusions in the INL and IPL at 2 and 5 months after injection. Arrows indicate cell body inclusions in the INL. Close up shows neurite-like inclusions in the IPL. Scale bar: 50 µm. B Quantification of the relative immunoreactivity intensity of pS129 staining in the IPL at 2 months and 5 months post injection. C Quantification of pS129-positive cell inclusions in the border of the INL at 2 months and 5 months post injection. D Representative images of colocalization of α-synuclein and pS129 in the INL. E Colocalization of pS129 and amacrine cell marker AP2. Scale bar: 20 µm. F – G Representative western blot images of α-synuclein in the soluble and insoluble fractions obtained from the whole retinal lysates at 2 months ( F ) and 5 months ( G ) after injection. Arrowheads show quantified monomer and truncated α-synuclein. H – K Quantification of monomer, aggregated (above 20 kDa) and truncated α-synuclein in the soluble ( H , J ) and insoluble fractions ( I , K ) at 2 months ( H , I ) and 5 months ( J , K ) after injection. Data in B - C and H – K are expressed as means ± s.e.m. (PBS, n = 5 mice; PFF, n = 5 mice; * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test and Two-way ANOVA)

Article Snippet: Later, samples were incubated overnight at room temperature with primary antibodies against α-synuclein (Syn1, 1:1000; BD Transduction, 610,787 clone 42/ α-synuclein, BD Biosciences, Franklin Lakes, NJ, USA), pS129 synuclein (1:500; Abcam, EP1536Y clone, ab51253), AP2 α (Invitrogen, 3B5 MA1-872, Waltham, MA, USA) in 5% goat serum.

Techniques: Injection, Staining, Marker, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils

doi: 10.1186/s40478-023-01575-0

Figure Lengend Snippet: Intravitreally injected α-synuclein causes brain α-synuclein accumulation 5 months after injection . A Representative images of pS129 immunostaining in the visual, perirhinal, and entorhinal cortices at 5 months after injection. B – D Quantification of pS129-positive cell puncta in the cortical areas. Numbers represent the total number of puncta in the indicated region from one coronal brain slice. Visual Cortex ( B ), Perirhinal Cortex ( C ) and Entorhinal Cortex ( D ). Brain areas were delimited using the Allen Atlas as reference. Scale bar: 100 µm. Data are expressed as means ± s.e.m. (PBS, n = 10; PFF, n = 10; * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test). E – F Representative western blots of α-synuclein in the rhinal cortex at 5 months post injection. F , G Quantification of α-synuclein monomer and aggregates (above 20 kDa) in the triton-x soluble E and insoluble F fractions. Data are expressed as means ± s.e.m, relative to control (PBS, n = 5; PFF, n = 5; * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test)

Article Snippet: Later, samples were incubated overnight at room temperature with primary antibodies against α-synuclein (Syn1, 1:1000; BD Transduction, 610,787 clone 42/ α-synuclein, BD Biosciences, Franklin Lakes, NJ, USA), pS129 synuclein (1:500; Abcam, EP1536Y clone, ab51253), AP2 α (Invitrogen, 3B5 MA1-872, Waltham, MA, USA) in 5% goat serum.

Techniques: Injection, Immunostaining, Slice Preparation, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils

doi: 10.1186/s40478-023-01575-0

Figure Lengend Snippet: Intravitreally injected α-synuclein causes the cortical inflammation 5 months after injection. A Representative images of microglia (Iba-1–positive cells) in the visual cortex, perirhinal cortex, and entorhinal cortex. B – D Quantification of the optical density of Iba-1 immunostaining in the cortical areas. E – G Microglial density in the visual, perirhinal and entorhinal cortices, normalized per area and expressed as microglia cell number per 10,000 µm 2 . (H) Representative images of TNF-α immunostaining in the cortical areas. (I-K) Quantification of TNF-α–positive cell counts in the cortical areas per 10,000 µm 2 . Scale bar: 100 µm. Data are expressed as means ± s.e.m. (PBS, n = 10; PFF, n = 10; * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t-test)

Article Snippet: Later, samples were incubated overnight at room temperature with primary antibodies against α-synuclein (Syn1, 1:1000; BD Transduction, 610,787 clone 42/ α-synuclein, BD Biosciences, Franklin Lakes, NJ, USA), pS129 synuclein (1:500; Abcam, EP1536Y clone, ab51253), AP2 α (Invitrogen, 3B5 MA1-872, Waltham, MA, USA) in 5% goat serum.

Techniques: Injection, Immunostaining

Journal: Acta Neuropathologica Communications

Article Title: Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils

doi: 10.1186/s40478-023-01575-0

Figure Lengend Snippet: α-synuclein propagation from the retina to cortical brain areas after intravitreal injection of PFFs. Injection of α-synuclein PFFs into the vitreous space caused deposition of pS129-positive neuritic (1) and cell body (2) inclusions in the IPL and INL, respectively. Accumulation of α-synuclein led to oxidative stress (3) and triggered apoptotic cell death of the RGCs (4), loss of tyrosine hydroxylase (5), and activation of optic nerve microglia (6). Injection of PFFs also resulted in aggregation of α-synuclein in the cortex (7) accompanied by inflammatory responses (8)

Article Snippet: Later, samples were incubated overnight at room temperature with primary antibodies against α-synuclein (Syn1, 1:1000; BD Transduction, 610,787 clone 42/ α-synuclein, BD Biosciences, Franklin Lakes, NJ, USA), pS129 synuclein (1:500; Abcam, EP1536Y clone, ab51253), AP2 α (Invitrogen, 3B5 MA1-872, Waltham, MA, USA) in 5% goat serum.

Techniques: Injection, Activation Assay

Journal: bioRxiv

Article Title: Impaired astrocyte-to-neuron cholesterol trafficking drives synaptic dysfunction in Rett syndrome

doi: 10.64898/2026.07.29.741426

Figure Lengend Snippet: ( A ) Representative images of primary branches from WT neurons treated for 24 hours with WT-ACM or KO-ACM, supplemented or not with cholesterol (0.1 μg/ml), and immunostained for Synapsin1/2 (Syn1/2; green), Shank2 (red) and Map2 (white). Scale bar=5 µm. ( B-D ) Violin plots indicate the median (dashed line) and 25 th and 75 th percentiles (dotted lines) of Synapsin1/2 (B), Shank2 (C) and colocalized puncta number (D). Values for puncta number are expressed as percentages with respect to neurons treated with WT-ACM. n=40/50. Statistical analysis was performed by 2-way Anova, followed by Sidak post-hoc test; *p<0.05, ***p<0.001.

Article Snippet: For the analyses of synaptic puncta density, neurons were stained with antibodies against Synapsin1/2 (#106006, Synaptic System; 1:500), Shank2 (#162 211, Synaptic System; 1:300) and Map2 (#8707, Cell Signalling; 1:1000).

Techniques:

Journal: bioRxiv

Article Title: Impaired astrocyte-to-neuron cholesterol trafficking drives synaptic dysfunction in Rett syndrome

doi: 10.64898/2026.07.29.741426

Figure Lengend Snippet: ( A ) Representative images of primary branches from WT and Mecp2 HET neurons, treated for 24 hours with cholesterol or with vehicle (0.1 μg/ml), and immunostained for Synapsin1/2 (Syn1/2; green), Shank2 (red) and Map2 (white). Scale bar=5 µm. ( B-D ) Violin plots indicate the median (dashed line) and 25 th and 75 th percentiles (dotted lines) of Synapsin1/2 (B), Shank2 (C) and colocalized puncta number (D). Values for puncta number are expressed as percentages with respect to untreated WT neurons. n=57/69. Statistical analysis was performed by 2-way Anova, followed by Sidak post-hoc test; **p<0.01, ***p<0.001. ( E ) Representative images of axon initial segment (AIS) stained with anti-AnkyrinG (AnkG; red). Scale bar=20 µm. ( F,G ) Violin plots indicate the median (dashed line) and 25 th and 75 th percentiles (dotted lines) of AIS length in WT and Mecp2 HET neurons (at DIV14) after cholesterol supplementation or treated with vehicle (F). The frequency distribution of values of AIS length is shown in (G). n=70/exp.groups. ( H ) Violin plots indicate the median (dashed line) and 25 th and 75 th percentiles (dotted lines) of the distance of AIS from the soma in WT and Mecp2 HET neurons (at DIV14) after cholesterol supplementation or treated with vehicle. n=70/exp.groups. *p<0.05, by 2-way Anova, followed by Sidak post-hoc test;

Article Snippet: For the analyses of synaptic puncta density, neurons were stained with antibodies against Synapsin1/2 (#106006, Synaptic System; 1:500), Shank2 (#162 211, Synaptic System; 1:300) and Map2 (#8707, Cell Signalling; 1:1000).

Techniques: Staining

Journal: Brain, behavior, and immunity

Article Title: Rats bred for low intrinsic aerobic exercise capacity link obesity with brain inflammation and reduced structural plasticity of the hippocampus.

doi: 10.1016/j.bbi.2021.06.017

Figure Lengend Snippet: Fig. 4. Older rats expressed less synaptic proteins (arbitrary units) in the hippocampus compared to young rats and the difference is more drastic in low-capacity Runner (LCR) rats than in high- capacity runner (HCR) rats. Western blot analyses of the expression of (A) synapsin (SYN-1) and (B) synaptophysin (SYP) in hippocampal homogenates of HCR and LCR rats at the age of 8 weeks (young) and 10 months (old). The membrane was cut horizontally to separate SYN-1 and SYP according to their molec ular weights, which are shown with the representative blot image. The graphs show the amount of the pro teins normalized to the total protein as analyzed from the stain-free blot. From the stain-free blot only the band of approximately 50 kDa is shown. The boxes and whiskers are the same as in Fig. 1. Mann-Whitney U test. * p ≤0.050, ** p ≤0.010 and *** p ≤0.001.

Article Snippet: Before that, the membranes were blocked for 1 h at RT with OdysseyR Blocking buffer (LI-COR 927–40000, Lincoln, NE, USA) and then incubated overnight at + 4 ◦C with primary antibodies against synaptic markers SYN-1 (ANR-014, Alomone labs, Jerusalem, Israel, 1:1000 dilution) and SYP (ANR-013, Alomone labs, Jerusalem, Israel, 1:1000), glial marker GFAP (ab7260, Abcam, Cambridge, UK, 1:5000 dilution) and microglial marker IBA1 (PA5-27436, ThermoFisher Scientific, Rockford, IL, USA, 1:700 dilution).

Techniques: Western Blot, Expressing, Membrane, Staining, MANN-WHITNEY