Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: Antibodies used for in vitro investigations.

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: In Vitro

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: Body mass evolution during 35 days of daily treatment with vehicle or AZP2006 post-lesion (A). Representative IHC images TH (red)/α-syn (green)/Iba1 (purple) (B). IHC analysis shows the total number of TH(+) neurons (C), area of α-syn accumulation in TH(+) neurons (D) and the number of Iba1(+) microglia in the SNpc (E) expressed as % of sham. Raw values for sham (i.e., 100%); TH(+) neurons = 65 ± 1.6; α-syn area/TH(+) neuron = 168 ± 9.4 µm 2 ; Iba1(+) cells = 53 ± 2.8. Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ## p < 0.01 or #### p < 0.0001 from sham; **p < 0.01; ***p < 0.001 and ****p < 0.0001 from α-syn+vehicle injury, (n = 7 mice / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A,E), the length of their neurite network (B,F), the area of cytoplasmic α-syn staining per TH(+) neuron (C,G) and the area of the post-synaptic marker PSD95 per TH(+) neuron (D and H), expressed as % of control (CTR). Raw values for CTR (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 27 ± 1.0; neurite network = 3540 ± 127 µm; α-syn area/TH(+) neuron = 201 ± 8.9 µm 2 ; PSD95 area/TH(+) neuron = 1829 ± 105 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ## p < 0.01; ### p < 0.001 or #### p < 0.0001 from CTR (control) or from anti-PGRN Ab and *p < 0.05 or ****p < 0.0001 from 6OHDA or from 6OHDA + anti-PGRN Ab; (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Staining, Marker, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A,E), the length of their neurite network (B,F), the area of cytoplasmic α-syn per TH(+) neuron (C,G) and lysosome burden as area of LAMP2(+) vesicles per TH(+) neuron (D and H), expressed as % of control (CTR). Raw values for MPP + & Rotenone CTR (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 18 ± 0.5 & 26 ± 1; neurite network = 889 ± 57 & 929 ± 24 µm; α-syn area/TH(+) neuron = 29 ± 2.1 & 28 ± 1 µm 2 ; ratio LAMP2/TH(+) = 10 ± 0.3 & 10 ± 0.5, respectively. Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. #### p < 0.0001 from CTR and *p < 0.05, **p < 0.01; ***p < 0.001 or ****p < 0.0001 from MPP + or Rotenone treatment, (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A), the length of their neurite network (B), the area of cytoplasmic α-syn staining per neuron (C), the area of LAMP2(+) staining per TH(+) neuron (D), the α-syn accumulation in lysosomes as α-syn/LAMP2 colocalization area (E), autophagosomes as LC3b(+) vesicles per TH(+) neuron (F), autolysosomes as LC3b(+) vesicles colocalized with LAMP2 area in TH(+) neurons (G), and functional lysosomes expressed as area of PhenoVue 577(+) staining per SH-SY5Y cell (H), expressed as % of control (CTR). Raw values for CTR (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 32 ± 1.3; neurite network = 1054 ± 60.6 µm; α-syn area/TH(+) neuron = 25.2 ± 2.8 µm 2 ; LAMP2 area/TH(+) neurons = 17.9 ± 1.5 µm 2 ; α-syn area/LAMP2/TH(+) neuron = 4.4 ± 0.3 µm 2 ; LC3b area/TH(+) neurons = 27 ± 1.7 µm 2 ; LAMP2/LC3b area/TH(+) neurons = 7.8 ± 0.5 µm 2 ; PhenoVue 577(+) staining/SH-SY5Y cell = 67 ± 3.5 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ### p < 0.001 or #### p < 0.0001 from CTR; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 from the α-syn condition, (n = 5-6).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Staining, Functional Assay, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A) and the length of their neurite network neurons (B) and microglia activation as area of OX-41(+) staining (C), expressed as % of control. Raw values for control (CTR) (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 81 ± 4.0; neurite network = 2332 ± 112 µm; OX-41(+) area/field = 1421 ± 67 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ### p < 0.001 or #### p < 0.0001 from CTR; **p < 0.01, ***p < 0.001 and ****p < 0.0001 from α-syn injury, (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Activation Assay, Staining, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis shows the total number of cells (A, D), the area of cytoplasmic α-syn staining per cell (B,E) and lysosomal burden as area of LAMP2(+) vesicles per cell (C,F), expressed as % of control (CTR = PGRN KO alone in D-F). Raw values for PGRN KO CTR (i.e., 100%); cells/image field = 140 ± 3.9; α-syn area/cell = 38.1 ± 2.8 µm 2 ; LAMP2(+) area/cell = 51 ± 2.8 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ## p < 0.01 or #### p < 0.0001 from CTR and *p < 0.05, **p < 0.01; ***p < 0.001 or ****p < 0.0001 from MPP + (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Staining, Control

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Chromatogram showing affinity purification of His-SUMO-EGFP-synphilin-1. Absorbance at 280 nm (y-axis, left) and the buffer employed expressed as percentage of elution buffer (y-axis, right). The fractions collected are highlighted in yellow. (B) Chromatogram showing size-exclusion (S200) purification of His-SUMO-EGFP-synphilin-1. The fractions collected are highlighted in yellow. (C) SDS-page gel showing all steps of EGFP-synphilin-1 purification (L: cell lysate, P: pellet of cleared lysate, S: supernatant of cleared lysate, FT: flow-through of affinity column, HE: His-eluate, SL: load to size exclusion column, PC: Pre-cleavage, CnH: SENP1 cleavage reaction, CH: reverse Ni-NTA flow-through). (D) SDS-page gel showing the recombinant, purified proteins used in the reconstitution assays: α-synuclein wild-type (left) and α-synuclein A140C (right). (E) Representative images of EGFP-synphilin-1 phase separation at different concentrations: 1 μM, 5 μM, 10 μM, and with increasing percentage of PEG 8,000 (0%, 0.5%, 1%, 3%, 5%, 10% w/v). Reconstitution experiments were performed at physiological salt concentrations: 25 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.5 mM TCEP. Samples were imaged at ambient temperature 5 min after incubation. (F) Reconstitution of 10 μM α-synuclein (chemically labeled with Alexa Fluor 568 or Alexa Fluor 647 (α-synuclein-568 and α-synuclein-647)) in varying percentages of PEG 8,000 (0%, 10%, 25% w/v). (G) Same as in F but using 100 μM α-synuclein. Scale bars, 10 μm. All data were consistent in three independent reconstitutions.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Affinity Purification, Purification, SDS Page, Affinity Column, Recombinant, Incubation, Labeling

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Purified EGFP-synphilin-1 (5 µM) forms condensates in 3% PEG 8,000 at room temperature. Scale bar, 10 µm. (B) Mapping of the combinations of EGFP-synphilin-1 and PEG 8000 where phase separation was observed (green circles) and not observed (grey circles). Phase separation was determined as the appearance of droplets at the coverslip surface 5 min after the beginning of the incubation. Gray circles: no condensates; light green indicate the formation of condensates smaller than 1 µm 2 ; dark green: condensates larger than 1 µm 2 . Each condition was assessed by three independent reconstitutions. (C) Reconstituted condensates containing EGFP-synphilin-1 (10 µM) and α-synuclein (20 µM) in 10% PEG 8,000 at ambient temperature 5 min (top) and 30 min (bottom) upon incubation. Note that both proteins colocalize in condensates after 5 min and demix to reach the core/shell structure after 30 min. Scale bar, 5 µm. (D) Line profiles of the highlighted selections in C. (E) Representative images of cells expressing EGFP-synphilin-1 and α-synuclein-BFP. Cell overview (left) and magnified representative images from indicated time points (right). Live-cell imaging indicates the growth of condensates over the period of 3 h, starting from the first mesoscale (>0,5 µm) puncta appearance. Note the demixing of α-synuclein and the evolution of the core-shell morphology. Scale bars, 10 µm (overview), 5 µm (magnified). (F) Quantification of the condensate radius over time for synphilin-1 (green line) and α-synuclein (magenta line), highlighting the larger size increase of the α-synuclein shell comparing to the synphilin-1 core. t shell marks the time at which the third phase appear, concomitant with the plateau of relative intensity. (G) Variation in the relative concentration within condensates compared to the cytoplasm for synphilin-1 (left) and α-synuclein (right). I cond and I cyt stand for the intensities within the condensate and the cytoplasm, respectively. t shell marks the time at which the third phase appear, concomitant with the plateau of relative intensity. Each gray trajectory denotes a distinct cell, with the colored line showcasing the mean and the shaded area indicating a range of two standard deviations. Data of ten condensates, each from a different cell, from three independent replicates. (H) Radial fluorescence intensity distributions (i.e., cross-sections averaged in all directions) from the central point of the condensate for synphilin-1 (left) and α-synuclein (right). The spatial coordinates of the condensate are normalized by its radius. The color code of the scheme reflects the time when each cut was taken. Note that as time progresses, the size of the condensate increases.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Purification, Incubation, Expressing, Live Cell Imaging, Morphology, Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Confocal image of EGFP-synphilin-1 ectopically expressed in HEK293 cells. Scale bar, 10 μm. (B) Representative fluorescence images of EGFP-synphilin-1 at 24 h (top) and 48 h (bottom) post-transfection in HEK293 cells before, during bleaching, and upon recovery after 60 s. Scale bars, 10 μm (overview), 3 μm (magnified). (C) Fluorescence recovery after bleaching of EGFP-synphilin-1 24- and 48-hours post-transfection in HEK293 cells. Values represent average ± standard error of the mean. Data are from three independent replicates. (D) Time-course images of cells expressing EGFP-synphilin-1, showing multiple fusions events leading to the formation of larger condensates. Scale bars, 10 μm. (E) Histogram showing the fraction of EGFP-synphilin-1 condensates exhibiting low circularity (≤0.89) and high circularity (≥0.9) at three time points (12 h, 24 h, and 48 h) post-transfection. Data from three independent replicates. (F) Co-expression of EGFP-synphilin-1 and α-synuclein-BFP results in demixing and accumulation of α-synuclein in the shell and synphilin-1 in the core. Right: magnification of EGFP-synphilin-1 and α-synuclein-BFP before and during bleaching, and upon recovery after 60 s (post-bleach). Scale bars, 10 μm (overview), 2 μm (magnified). (G) Fluorescence recovery after photobleaching of EGFP-synphilin-1 (green) and α-synuclein-BFP (magenta) at the rim of condensates. Values are represented as average ± standard error of the mean. The experiment was performed in three independent replicates, with at least ten condensates analyzed.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Confocal, Fluorescence, Transfection, Expressing

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Schematic of the volume intersected by the focus layer as a function of the position of the droplet for different regimes of the droplet radius. The dashed part of the droplet corresponds to the portion of the condensate that is not included in the focus layer. We assume that the droplet can be at any depth relative to the focus layer, from z = 0 to z = W, equiprobably, to compute the average volume. The volume is computed by summing infinitesimal slices of the sphere from θ 0 to θ 1 . (B) Change in the relative concentration of proteins (synphilin-1, left; α-synuclein, right) over time. Individual cells are represented by the gray curves, while the blue line denotes the average. The shaded gray area indicates a range of two standard deviations. (C) Representative fits of the radial intensity profiles of the condensate boundary using the difference of two sigmoid functions to extract droplet radius measurements at selected time points.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Orthoslice (virtual section) from a representative tomogram of a cell expressing EGFP-synphilin-1. Left, single slice of an X-ray tomogram; right, highlighted surface-rendered synphilin-1 condensate (cyan). Scale bar, 3 µm. (B) Surface-rendered view of the cell in A , highlighting all synphilin-1 condensates inside the cell. ( C) Virtual section of a synphilin-1 condensate. Scale bar, 2 µm. (D) Line profile through the equatorial plane indicates a homogeneous linear absorption coefficient (LAC) of contents. (E) Virtual section from representative tomogram of a cell expressing LBL condensates (EGFP-synphilin-1 and α-synuclein-BFP). Left, single orthoslice of an X-ray tomogram; right, highlighted surface-rendered LBLs (cyan). Scale bar, 3 µm. (F) Surface-rendered cell in E , highlighting all LBLs inside the cell. (G) Virtual section of two LBLs. Scale bar, 2 µm. (H) Line profile through the equatorial plane showing the changes in LAC. Note the distinct absorption pattern at the core (yellow) and shell (magenta) of the condensate, indicating distinct material properties of these two regions within a condensate. (I) Left: orthoslice of a pair of LBLs in close proximity but remaining non-fused (i.e., the absence of content mixing); middle: 2D histogram of LAC values as a function of radial distance; right: representative interpolation of the LBL pair. (J) The same as in I but for LBLs that undergo fusion-fission (i.e., the presence of content mixing).

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Expressing

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Left: orthoslice from a representative tomogram of a cell expressing LBLs (EGFP-synphilin-1 and α-synuclein-BFP). Red arrows indicate LBLs. Right: highlighted surface-rendered LBLs (cyan). Scale bar, 2 μm. (B) Surface-rendered LBLs of a selected region in A (red arrowheads) captures fusing LBLs. Scale bar, 2 μm. (C) Quantification of linear absorption coefficients (LACs) for synphilin-1 condensates and LBLs suggests a change in the material properties (i.e., an increase in density of carbon-rich molecules) in condensates over time; three independent experiments. p ≤ 0.0001, unpaired t -test.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Expressing

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Left: confocal images of HEK293 cells expressing EGFP-synphilin-1, before and after the addition of 5% DMEM in H 2 O. Right: Fluorescence intensity profiles before (solid line) and after (dotted line) exchange of media. (B) Same as in A but before and upon perfusion of 300 mM NaCl solution (hypertonic solution). (C) Left: confocal images of cells expressing EGFP-synphilin-1 and α-synuclein-BFP, before and after exchange of live cell imaging media to the hypotonic solution (5% DMEM in H 2 O); Right: Line profiles for synphilin-1 (green) and α-synuclein (magenta). Fluorescence intensity profiles in conditions before (solid lines) and after (dotted lines) treatment. (D) Same as in C but before and after perfusion with hypertonic solution (300 mM NaCl in H 2 O). Scale bars, 10 μm. All experimental conditions were probed in three independent replicates. Yellow lines indicate lines along which fluorescence intensity profiles were obtained.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Confocal, Expressing, Fluorescence, Live Cell Imaging

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Cluster map of NARDINI+ z-score vectors highlights distinctive molecular grammar features of synphilin-1 IDR and α-synuclein variants. (B) Schematic showing synphilin-1 IDR mean hydropathy score (top) and mean fraction of aliphatic residues minus fraction of charged residues (bottom). (C) Same schematic as in B for α-synuclein. (D) Representative images (left) of cells 24 h after co-transfection with a truncated version of α-synuclein-BFP (deleted residues Δ37-94, so-called ΔF; top scheme) and EGFP-synphilin-1 and cumulative line profile (right), indicating the demixing of α-synuclein ΔF from synphilin-1. Scale bar, 10 µm. (E) Representative images (left) of cells 24 h after co-transfection with a truncated version of α-synuclein-BFP (deleted residues Δ96-140, so-called ΔC; top scheme) and EGFP-synphilin-1 and cumulative line profile (right) indicating the demixing of α-synuclein ΔC from synphilin-1. Scale bar, 10 µm. (F) Representative images (left) of cells 24 h after co-transfection with α-synuclein-BFP A30P and EGFP-synphilin-1. and cumulative line profile (right) indicating the demixing of α-synuclein A30P from synphilin-1. Scale bar, 10 µm (G) Representative images (left) of cells 24 h after co-transfection with S129E phosphomimetic version of α-synuclein-BFP and EGFP-synphilin-1 and cumulative line profile (right) indicating the demixing of α-synuclein S129E from synphilin-1. Scale bar, 10 µm. (H) Representative images of HEK293 cells expressing early-stage LBLs (top; EGFP-synphilin-1 (green) and α-synuclein-BFP (magenta) are fully mixed), intermediate (middle), and late-stage LBLs (bottom; EGFP-synphilin-1 and α-synuclein-BFP form core-shell morphology) and immunostained for pS129 α-synuclein (blue). Note that pS129 α-synuclein starts accumulating at the periphery of condensates at an earlier stage when the total, unphosphorylated α-synuclein still remains enriched across the entire condensate. Scale bar, 10 μm. (I) The cumulative line profiles of conditions in H showing progressive pS129 and total α-synuclein demixing. (J) Schematic representation recapitulating the maturation of LBLs. At early stages, all components are intermixed; as the condensates mature, the internal rearrangements favor the demixing of α-synuclein in the shell and the enrichment of pS129 α- synuclein. The data in D-G and I represent an average ± s.e.m. of ten condensates each from a different cell from three independent replicates/experiments.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Cotransfection, Expressing, Morphology

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Agarose gel electrophoresis for PCR products of junctions between the integration cassette and the genomic part of AAVS1 integration locus in the AK-11 cell line. (B) Western blots of α-synuclein expression level in the AK-11 cell line and after transient transfection in HEK293 cells. Lysates were collected 24 hours after induction or transfection respectively and immunoblotted against α-synuclein. Dashed lines indicate spliced irrelevant bands. (C) Representative confocal image of AK-11 cell line in culture. Scale bar, 50 µm. (D) Changes in the fraction of expressing cells over passages. AK-11 cells are stable throughout time. Data from at least 1300 cells across three independent replicates for each timepoint. (E) Representative images of AK-11 cells, transfected with mCherry-synapsin-1 in AK-11 simultaneously with the induction of α-synuclein expression. Scale bar, 10 µm. (F) Fraction of transfected cells that contain condensates after mCherry-synapsin-1 transfection in AK-11 and mCherry-synapsin-1 and α-synuclein-BFP co-transfection in HEK293 cells. Data from at least 600 cells across three independent replicates. (F) Synapsin-1 (left) and α-synuclein (right) enrichment in condensates imaged 24 hours after mCherry-synapsin-1 transfection and induction in AK-11 and after double transfection in HEK293 cells. (H) Synapsin-1/α-synuclein condensates size frequency distribution 24 hours after mCherry-synapsin-1 transfection and induction in AK-11 and after double transfection in HEK293 cells. Lines represent kernel distribution estimate curves. (I) Number of condensates per cell 24 hours after mCherry-synapsin-1 transfection and induction in AK-11 and after double transfection in HEK293 cells. In G and I the box stretches from the 25th to 75th percentile, dots represent individual data points, the central line shows the median, and whiskers represent the min and max values. Data in G , H and I were collected from at least 15 cells across three independent replicates.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Agarose Gel Electrophoresis, Western Blot, Expressing, Transfection, Confocal, Cotransfection

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) The proteome of LBLs-expressing cells is altered across a wide range of subcellular compartments. Numbers represent the number of significantly changed proteins (left) and total number of detected proteins (right) for each compartment. Protein subcellular localization was determined by Gene Ontology: Cellular Compartment analysis. Significance threshold: q≥0.05, |log2FC|≥0.3. (B) The volcano plot demonstrates changes in cytoskeletal proteins expression in LBLs-expressing cells. Magenta dots represent significantly changed cytoskeleton-related proteins. Significance threshold: q≥0.05, |log2FC|≥0.3. (C) Live-cell imaging demonstrates the enrichment of SiR-Actin inside the condensates (green, EGFP-synphilin-1; magenta, SiR-Actin; α-synuclein is not shown for clarity). Left, whole-cell overview; right, magnified condensate. Scale bar, 10 µm. (D) Live-cell imaging indicates no accumulation of SiR-Tubulin inside the condensates (green, EGFP-synphilin-1; magenta, SiR-Tubulin; α-synuclein is not shown for clarity). Left, whole-cell overview; right, magnified condensate. Scale bar, 3 µm. (E) Quantification of SiR-Actin (top) and SiR-Tubulin (bottom) signal inside the condensates (three independent experiments, fifteen condensates analyzed for each reporter). Note the enrichment of actin signal inside the condensate and the steady levels (i.e., the absence) of SiR-Tubulin signal. (F) Latrunculin A treatment inhibits formation of LBLs in AK-11 cells, expressing EGFP-synphilin-1 and α- synuclein-Halo in comparison with untreated controls, as represented by the fraction of LBLs-containing cells. Data from at least 2000 cells across at least three independent replicates for each condition. Error bars represent SD; dots represent individual replicates. Significance was tested using a Student’s t-test; asterisks, significance: ****p<0.0001.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Expressing, Analysis, Cytoskeletal, Live Cell Imaging, Comparison

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Images of two representative condensates, each from a separate experiment, of cells expressing LBLs (green, EGFP-synphilin-1) and incubated with MitoTracker-647 (orange) and SPY555-actin (white in overlay) and corresponding circular line profiles. Orange lines indicate the line along which circular profile was obtained in the respective image. Scale bars, 3 µm. (B) Representative images of cells expressing EGFP-synphilin-1 incubated with FM4-64 (top), LysoTracker-647 (middle) and MitoTracker-647 (bottom) for 30 min before imaging. Blue arrows indicate organelles in the vicinity of LBLs. Scale bars, 10 μm. (C) Representative images of cells expressing EGFP-synphilin-1 and α- synuclein-BFP incubated with FM4-64 (top) and LysoTracker-647 (bottom) for 30 min before imaging. Blue arrows indicate organelles in the vicinity of LBLs. Scale bars, 10 μm. All experimental conditions were performed in three independent replicates.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Expressing, Incubation, Imaging

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Live-cell confocal microscopy images of HEK293 cells expressing EGFP-synphilin-1 and α-synuclein-BFP, 24 h post-transfection and incubated with MitoTracker-647. Scale bar, 10 µm. (B) Representative electron micrograph of cryo-fixed HEK293 cells expressing LBLs (left) and magnified tomogram slice with its 3D rendered volume (right; magenta: ER, yellow: mitochondria, blue: LBL). Note accumulation of organelles at the condensate interface. Dashed line indicates region used for electron tomography. N indicates nucleus; M, mitochondria; ER, endoplasmic reticulum; *, LBL. Scale bar, 1 µm (micrograph), 200 nm (tomogram). (C) SXT orthoslice of a representative cell expressing EGFP-synphilin-1. Asterisk indicates synphilin-1 condensate. Scale bar, 2 µm. (D) SXT orthoslice of a representative cell expressing LBLs (EGFP-synphilin-1 and α-synuclein-BFP). Asterisk indicates an LBL. Scale bar, 2 µm. (E) Surface rendering of the cell in C, indicating the reconstructed mitochondrial network (yellow) around synphilin condensate (cyan). (F) Surface rendering of the cell in D, indicating the reconstructed mitochondrial network (yellow) around an LBL (cyan). Note the accumulation of mitochondria at the interface of an LBL. (G) SXT orthoslice of a representative tomogram of a cell expressing EGFP-synphilin-1 and α-synuclein-BFP demonstrates the accumulation of mitochondria at the interface of Lewy body-like condensates. Cyan, surface-rendered LBL; yellow, surface-rendered mitochondria network. Scale bar, 3 µm. (H) Magnified region indicates contacts between mitochondria and LBLs (red arrowheads). Scale bar, 3 µm. (I) Surface-to-volume ratios of mitochondria rendered from the SXT obtained from untransfected cells (blue), cells expressing synphilin-1 (green), LBLs-containing cells (magenta) and cells expressing synphilin-1 and α-synuclein and treated with Latrunculin A. Data are from three independent experiments. Significance was tested using one-way ANOVA test; ns, non-significant; asterisks, significance: ****, p < 0.0001.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Confocal, Microscopy, Expressing, Transfection, Incubation, Tomography

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Representative overview of HEK cells, expressing EGFP-synphilin-1 and α -synuclein-BFP fixed on EM grid. White dashed line indicates outline of zoomed region. Scale bar, 20 μm. (B) Magnified CLEM image of individual cell from A . Scale bar, 5 μm. (C) Transmission electron microscopy image for the cell from B . Red arrows indicate organelles in close proximity of LBL. N, nucleus; *, LBL. Scale bar, 5 μm.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Expressing, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Representative images of wild-type murine hippocampal neurons in culture expressing early-stage (top; EGFP-synphilin-1 (green) and α-synuclein-BFP (magenta) are fully mixed), and late-stage LBLs (bottom; EGFP-synphilin-1 and α-synuclein-BFP form core-shell morphology). White dashed squares delineate magnified regions; white dotted lines represent neurites. Scale bars, 20 µm (overview), 5 µm (magnified). (B) Cumulative line profiles for conditions in A . Line profiles were collected from 10 condensates across 3 biological replicates, each from a separate cell. Bold line represents mean value, shaded area represents SD. (C) LatA treatment inhibits LBLs formation in neurons, as represented by the fraction of LBL-containing cells. Data from at least 300 cells across three independent replicates for each condition. Error bars represent SD; dots represent individual replicates; lines connect values for DMSO control and LatA treatment from the same biological replicate. Significance was tested using a paired Student’s t-test; asterisks, significance: *, p<0.05. (D) Immunohistochemistry on paraffinized human brain tissues from individuals with no Lewy body pathology (control), stained against synphilin-1 (green), pS129 α-synuclein (blue), and VDAC channel on mitochondria (orange). Note the absence of LBs. Scale bars, 10 µm. (E) Representative examples of different stages of LB pathology in the substantia nigra and hippocampus from individuals with LB pathology stained against synphilin-1 (green), pS129 α-synuclein (blue), and VDAC channel on mitochondria (orange). Note the accumulation of mitochondria fragments at the interface of LBs in the merged images. In total, 11 LBs were analyzed from three individuals, 9 of which were positive for synphilin-1. Scale bars, 10 µm (overview), 3 µm (magnified). (F) Fluorescence intensity profiles across the interface of LBs shown in E. (G) Radial line profiles across the LBs from individual LBs shown in E , demonstrating the distribution density of mitochondria at the interface of LBs.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Expressing, Morphology, Control, Immunohistochemistry, Pathology, Staining, Fluorescence

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Schematic illustrating the experiment setup. α-synuclein PFFs, HEK293 cells containing LBLs, and a histological section containing a LB from the substantia nigra of a PD patient were raster-scanned with a focused X-ray beam (beam diameter ≈ 250 nm, photon energy = 15 keV), recording a 2D scattering pattern in each scan point by a photon-counting pixel detector. (B) X-ray dark-field map of dried α-synuclein PFFs. The intensity was integrated between q = [0.4, 1.9] Å -1 . Scale bar, 10 µm. (C) Average scattering profile of α-synuclein PFFs within the ROI selected in B . The sum of three Gaussians is fitted to the data. (D) Optical micrograph of freeze-dried HEK293 cells expressing EGFP-synphilin-1 and α-synuclein-BFP (composite image of the bright-field, EGFP, and BFP channels). An LBL is visible in the lower-right cell. Scale bar, 10 µm. (E) X-ray dark-field map of the sample from D . The intensity was integrated between q = [0.4, 1.9] Å -1 . Scale bar, 10 µm. (F) Average scattering profile of the LBL from E . The sum of three Gaussians is fitted to the data. (G) Optical micrographs of a hematoxylin & eosin-stained histological section from the substantia nigra of a PD patient containing a LB. The section was air-dried immediately before the X-ray measurements, resulting in some cracks. The inset shows the same LB before drying. Scale bar, 25 µm (overview), 10 µm (inset). (H) X-ray dark-field map of the sample from G, showing the integrated intensity between q = [0.4, 1.9] Å-1. Scale bar, 25 µm (I) Average scattering profile of the LB shown in H . The sum of three Gaussians is fitted to the data. A description of the curve-fitting process and parameters of Gaussians for each curve are provided in the Methods section and Supplementary Tables S4-6. (J) Conceptual schematic summary of the study. Normally, α-synuclein is a soluble presynaptic protein with a physiological role. However, in pathology it is found in aggregates in neuronal soma and neurites. Our study provides a platform that faithfully recapitulates early stages of LB formation through phase separation, including fibrillization of α-synuclein and sequestration of membrane-bound organelles.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Counting, Expressing, Staining, Pathology, Membrane

Journal: bioRxiv

Article Title: Reverse engineering Lewy bodies suggests α-synuclein pathology as condensatopathy

doi: 10.64898/2026.10.09.757996

Figure Lengend Snippet: (A) Immunostaining for p62 in HEK293 cells expressing EGFP-synphilin-1 and α-synuclein-BFP in early stages with fully mixed signal of LBLs (top) and the mature LBLs with core-shell architecture (bottom). Note the enrichment of p62 puncta around LBLs in both cases. Scale bars, 10 µm (overview), 3 µm (3D-reconstruction). (B) Immunostaining for p62 in murine primary hippocampal neurons expressing EGFP-synphilin-1 and α-synuclein-BFP. Yellow arrows indicate p62 bodies in contact with LBLs. Scale bar, 10 µm.

Article Snippet: The pET28a expression vector was created by subcloning α-synuclein (Addgene #51437) with restriction enzyme sites Nco I- Not I into the pET28a vector.

Techniques: Immunostaining, Expressing

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: Study and experimental flow chart. This Figure illustrates the overall experimental design and workflow of the study. The study integrates bioinformatic analyses with in vitro and in vivo experimental validations. Bioinformatic approaches included the single-cell transcriptome analysis of Parkinson’s disease (PD) patient midbrains to identify differentially expressed genes (DEGs) in dopaminergic neurons (DaNs) and cell type alterations, molecular docking between clemastine (CLE) and 22,618 proteins to identify potential targets, and correlation analysis between Braak staging and autophagy gene expression. The intersection of CLE targets and PD-related DEGs was used to predict the CLE’s mechanism of action, focusing on the AMPK/mTOR pathway and autophagy. These predictions were tested experimentally using an MPP + -induced PC12 cell model and a chronic MPTP-induced mouse model of PD. Endpoints assessed included protein expression (western blot and immunofluorescence), autophagic flux, α-synuclein (α-syn) and phosphorylated α-syn (p-α-syn) levels, dopaminergic neuron survival, motor function (rotarod test), and non-motor function (fecal pellet analysis). The integrative approach aims to demonstrate that CLE alleviates PD pathology by activating the AMPK/mTOR pathway, enhancing autophagic flux, and promoting the clearance of pathological α-syn. PD, Parkinson’s disease; CLE, Clemastine; MPTP, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP + , 1-methyl-4-phenylpyridinium; DEGs, differentially expressed genes; DaNs, dopaminergic neurons; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; α-syn, α-synuclein; p-α-syn: phosphorylated α-synuclein.

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: In Vitro, In Vivo, Single Cell, Analysis, Docking Assay, Gene Expression, Expressing, Western Blot, Immunofluorescence, Pathology

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: CLE reduces α-synuclein protein levels and improves cell survival, effects inhibited by dorsomorphin (DOR). (A) Western blot (WB) and (B) The quantification of α-synuclein (α-syn) protein levels in PC12 cells from CTRL, MPP + , CLE, and MPP + +CLE groups. GAPDH is shown as a loading control. (C) Cell survival rate in CTRL, MPP + , and MPP + +CLE groups. (D, G) The WB analysis of p-AMPK (Thr172), t-AMPK, p-mTOR (Ser2448), and t-mTOR protein levels in CTRL, DOR, CLE, and DOR+CLE groups. (E, F, H) The Quantification of the indicated protein ratios. Data are presented as mean ± SEM. Tukey’s multiple comparison test was performed after one-way ANOVA. * P < 0.05, ** P < 0.01 *** P < 0.001; ns, not significant. CLE, clemastine; α-syn, α-synuclein; CTRL, control; WB, western blot; DOR, dorsomorphin; ANOVA, analysis of variance; SEM, standard error of the mean. n = 5 independent experiments.

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: Western Blot, Control, Analysis, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: CLE increases AMPK phosphorylation, decreases mTOR phosphorylation at Ser2448, enhances autophagy, and reduces α-synuclein levels in the mouse midbrain. (A) The WB analysis of p-AMPK (Thr172), t-AMPK, p-mTOR (Ser2448), and t-mTOR protein levels in the midbrain of mice from CTRL, MPTP, and MPTP+CLE groups. GAPDH is shown as a loading control. (B) The quantification of p-mTOR/t-mTOR levels. (C) The quantification of p-AMPK/t-AMPK levels. (D–F) The WB analysis of p62, LC3-I/II, and α-syn protein levels. (G–I) The quantification of p62, LC3-II/LC3-I ratio, and α-syn protein levels. Data are presented as mean ± SEM. Tukey’s multiple comparison test was performed after one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. CLE, clemastine; α-syn, α-synuclein; CTRL, control; WB, western blot; ANOVA, analysis of variance; SEM, standard error of the mean. n = 5 independent experiments.

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: Phospho-proteomics, Analysis, Control, Comparison, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: CLE increases LC3 and reduces phosphorylated α-synuclein in dopaminergic neurons (DaNs) of MPTP-treated mice. (A) Immunofluorescence staining for TH (red, marker for DaNs), LC3 (green), and DAPI (blue) in the substantia nigra (SN) of mice from CTRL, MPTP, and MPTP+CLE groups. Scale bar: 2 μm. (B) The quantification of the relative number of LC3 puncta in DaNs. (C) Immunofluorescence staining for TH (red), p-α-syn (Ser129, green), and DAPI (blue) in the SN. Scale bar: 5 μm. (D) The quantification of the relative fluorescence intensity of p-α-syn (Ser129) in DaNs. Data are presented as mean ± SEM (N = 21 per group). Tukey’s multiple comparison test was performed after one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. CLE, clemastine; DaNs, dopaminergic neurons; p-α-syn, phosphorylated α-synuclein (Ser129); SN, substantia nigra; CTRL, control; ANOVA, analysis of variance; SEM, standard error of the mean. n = 21 images per group (7 images from 3 mice per group).

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Comparison, Control, Analysis