Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A M83 mice underwent stereotaxic surgery for inoculation of aSyn PFF-1, aSyn PFF-2 or PBS control into the right dorsal striatum. At 8 weeks post injection, they subsequently underwent a motor test battery, including assessments of motor strength, motor coordination and gait. Following completion, they were assessed for the cognitive ability to acquire stimulus-response associations, with the ‘Visuomotor Conditional Learning’ at 9-12 weeks post injection. At 16 weeks post-injection, they repeated the motor test battery, and then were processed for pathology and biochemistry. B Brain homogenates from aSyn PFF-1 and PFF-2 inoculated mice both displayed resistance to proteinase K but exhibited variable banding patterns upon digestion, as assessed by Western Blot using an antibody for total aSyn. C, D Furthermore, the same brain homogenates were evaluated by dotblots using an antibody with high affinity for aggregated aSyn (chBIIB054), revealing that while both fibril types exhibited significant binding, aSyn PFF-2 brain homogenates presented with a stronger signal (Welch’s ANOVA test, W 2, 5.34 = 7.03, p < 0.05, in which Welch-corrected unpaired t-tests revealed to be driven by a significant difference between PBS control and PFF-2 (p < 0.05). Graphics made with Biorender.com.

Article Snippet: M83 hemizygous mice (M83 + /− mice; B6; C3H-Tg[SNCA]83Vle/J) were obtained from Jackson Laboratory (004479) and maintained in-house on a C57BL/C3H background.

Techniques: Control, Injection, Battery, Western Blot, Binding Assay

Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A Schematic of Visuomotor Conditional Learning (VMCL) task. Cognitive assessments were conducted within touchscreen systems equipped with a touch-sensitive screen, a reward magazine attached to a reward pump for delivery of strawberry milkshake liquid reward and ABET cognition software ( above ). VMCL was employed to evaluate the acquisition of stimulus-response (S-R) contingencies, with subjects learning the conditional rule: “if visual stimulus A is presented, make motor response to the right-flanking window; if visual stimulus B is presented, make motor response to the flight-flanking window” ( below ). All subjects underwent VMCL testing for 20 sessions, 5-7 sessions per week. B M83 mice inoculated with aSyn PFF-1 and PFF-2 were significantly impaired at acquiring VMCL, as demonstrated by lower percent correct responses relative to PBS-inoculated mice (2-way RM ANOVA, Group x Session: main effect of group (F 2,60=10.53 , p < 0.001), main effect of session (F 2.686, 161.2=10.53 , p < 0.0001) and significant interaction (F 8,240=3.131 , p < 0.01), in which Šídák’s multiple comparisons revealed to be driven by a significant difference between PBS control and PFF-1 in sessions 2 (adj. p < 0.05), 4 (adj. p < 0.05) and 5 (adj. p < 0.05), and a significant difference between PBS control and PFF-2 in sessions 2 (adj. p < 0.01), 3 (adj. p < 0.001), 4 (adj. p < 0.01), and 5 (adj. p < 0.01)). C While all groups began at chance in Block 1 (1-way ANOVA, p > 0.05), (D) both aSyn-inoculated groups were significantly impaired relative to controls in Block 5 (1-way ANOVA, F 8.764 , p < 0.001, in which Šídák’s multiple comparisons revealed to be driven by a significant difference between PBS controls and PFF-1 (adj. p < 0.05), and between PBS controls PFF-2 (adj. p < 0.001)). E No significant difference was found in the percentage of missed trials across groups (RM Mixed-Effects Model, Group x Session: main effect of session (F 2.050,118.4= 20.55 , p < 0.0001), but no main effect of group or interaction (p > 0.05)), (F) but aSyn PFF-1 and PFF-2 mice exhibited a greater number of correction trials (2-way RM ANOVA, Group x Session: main effect of group (F 2,60= 6.008 , p < 0.01), main effect of session (F 3.240,194.4= 66.22 , p < 0.0001) but no interaction (p > 0.05)), and (G) an elevated perseveration index compared to controls (RM Mixed-Effects Model, Group x Session: main effect of group (F 2,60=4.648 , p < 0.05), main effect of session (F 3.191,171.5=24.48 , p < 0.0001), but no interaction (p > 0.05)). Furthermore, comparing task latencies, no significant difference was found in the latency to make correct choices (H) (2-way RM ANOVA: Group x Session: p > 0.05), but aSyn PFF-1 and PFF-2 mice took significantly longer to make incorrect choices (I) across VMCL acquisition compared to PBS controls (2-way RM ANOVA: Group x Session: main effect of group (F 2,60=5.384 , p < 0.01), main effect of session (F 3.202,192.1= 62.37 , p < 0.0001), and significant interaction (F 8,240=1.985 , p < 0.05), in which Šídák’s multiple comparisons revealed to be driven by a significant difference between PBS control and PFF-1 in sessions 2 (adj. p < 0.01), 3 (adj. p < 0.01) and 5 (adj. p < 0.05), and a significant difference between PBS control and PFF-2 in sessions 3 (adj. p < 0.05) and 5 (adj. p < 0.05)). No significant difference was found for the latency to collect rewards (J) (2-way RM ANOVA, Group x Session: main effect of session (F 2.275,135.9=17.67 , p < 0.0001), but no main effect of group or interaction (p > 0.05)). Data presented as Mean + SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Graphics made with Biorender.com.

Article Snippet: M83 hemizygous mice (M83 + /− mice; B6; C3H-Tg[SNCA]83Vle/J) were obtained from Jackson Laboratory (004479) and maintained in-house on a C57BL/C3H background.

Techniques: Software, Control, Blocking Assay

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of intracerebral inoculation experiments in M83 +/- mice. b ) Silver-stained SDS-PAGE of the detergent-insoluble fraction following PK digestion of monomeric (mono) recombinant A53T-mutant α-syn and the PFF samples used for inoculation experiments. c ) Kaplan-Meier survival curves for M83 +/- mice inoculated with monomeric A53T-mutant α-syn (n = 9), A53T 1 PFFs (n = 23), or A53T 2 PFFs (n = 25). For the PFF inoculations, data from the three replicate experiments for each polymorph is pooled. Statistical significance was determined using the Log-rank test. d ) Immunoblot of detergent-insoluble PSyn species in brain extracts from M83 +/- mice at the indicated DPI with either monomeric A53T α-syn (asymptomatic mice) or A53T PFFs (symptomatic mice). Two representative mice for each inoculum are shown. e ) Quantification of detergent-insoluble PSyn species in brain extracts from symptomatic M83 +/- mice inoculated with either A53T 1 PFFs (n = 18) or A53T 2 PFFs (n = 25). f ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from symptomatic M83 +/- mice at the indicated DPI with A53T 1 PFFs or A53T 2 PFFs. Two representative mice for each inoculum are shown. g ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from symptomatic M83 +/- mice at the indicated DPI with individual replicates of A53T 1 PFFs or A53T 2 PFFs. h ) Representative immunoblots for residual insoluble TL-resistant α-syn species following treatment of brain extracts from symptomatic M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs with increasing concentrations of GdnHCl. i ) [GdnHCl] 50 values for TL-resistant α-syn species in symptomatic M83 +/- mice inoculated with either A53T 1 or A53T 2 PFFs (n = 9 each, 3 mice from each replicate experiment). In panels e and i, graphs display mean ± s.e.m. and statistical significance was assessed using a Mann-Whitney test. In panels b, d, f, g, and h, molecular weight markers indicate kDa.

Article Snippet: Homozygous M83 (M83 +/+ ) mice (stock number: 004479), which use the mouse prion protein promoter to overexpress human A53T mutant α-synuclein on a mixed C57BL/6 and C3H background [ ], and non-transgenic B6C3F1 mice (stock number: 100010) were purchased from The Jackson Laboratory.

Techniques: Staining, SDS Page, Recombinant, Mutagenesis, Western Blot, MANN-WHITNEY, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Quantification of the area covered by PSyn staining in the indicated brain regions from symptomatic M83 +/- mice injected with A53T 1 PFFs (n = 18) or A53T 2 PFFs (n = 25). b ) Representative images of cortical PSyn deposits in brain sections from M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs. c ) Quantification of the number of cortical PSyn deposits in brain sections from M83 +/- mice inoculated with A53T 1 (n = 18) or A53T 2 (n = 25) PFFs. d ) Representative images of hippocampal PSyn deposits (CA1 and dentate gyrus regions) in brain sections from M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs. e ) Quantification of the number of PSyn deposits in the CA1 and dentate gyrus regions from M83 +/- mice inoculated with A53T 1 (n = 18) or A53T 2 (n = 25) PFFs. f ) Representative images of ring-like PSyn deposits in the midbrain and hypothalamus from M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs. g ) Quantification of ring-like vs. Lewy body (LB)-like PSyn deposits in the midbrains of M83 +/- mice inoculated with A53T 1 (n = 18) or A53T 2 (n = 25) PFFs. In panels a, c, e, and g, the graphs display mean ± s.e.m. In panels a, c, and e, statistical significance was assessed by a Mann-Whitney test. In panel g, statistical significance was assessed by two-way ANOVA. In panels b, d, and f, the scale bar indicates 20 µm and applies to all images.

Article Snippet: Homozygous M83 (M83 +/+ ) mice (stock number: 004479), which use the mouse prion protein promoter to overexpress human A53T mutant α-synuclein on a mixed C57BL/6 and C3H background [ ], and non-transgenic B6C3F1 mice (stock number: 100010) were purchased from The Jackson Laboratory.

Techniques: Staining, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Kaplan-Meier survival curve for M83 +/+ mice (n = 32). b ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from spontaneously ill M83 +/+ mice at the indicated ages. Based on the patterns of TL- and PK-resistant species, each mouse was assigned as either subtype X, Y, or Z. Two representative mice for each subtype are shown. Brain extract from a young asymptomatic M83 +/+ mouse is included as a negative control. Molecular weight markers indicate kDa. c ) Relative frequencies of M83 +/+ X , M83 +/+ Y , and M83 +/+ Z subtypes across the 32 observed M83 +/+ mice. d ) Age of spontaneous motor dysfunction onset in M83 +/+ mice classified as M83 +/+ X (n = 8), M83 +/+ Y (n = 20), or M83 +/+ Z (n = 4). Male mice are indicated by open circles and female mice by closed circles. e ) Representative images of cortical PSyn deposits in brain sections from spontaneously sick mice exhibiting the M83 +/+ X , M83 +/+ Y , or M83 +/+ Z subtype. f ) Quantification of the number of cortical PSyn deposits in brain sections from spontaneously sick mice displaying the M83 +/+ X (n = 8), M83 +/+ Y (n = 20), or M83 +/+ Z (n = 4) subtype. g ) Representative images of ring-like and Lewy body (LB)-like PSyn deposits in the midbrain and hypothalamus from spontaneously ill M83 +/+ mice. h ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of spontaneously ill M83 +/+ mice exhibiting the M83 +/+ X (n = 8), M83 +/+ Y (n = 20), or M83 +/+ Z (n = 4) subtype. All graphs display mean ± s.e.m. In panels d and f, statistical significance was assessed by a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. In panel h, statistical significance was assessed by two-way ANOVA. In panels e and g, the scale bar indicates 20 µm and applies to all images.

Article Snippet: Homozygous M83 (M83 +/+ ) mice (stock number: 004479), which use the mouse prion protein promoter to overexpress human A53T mutant α-synuclein on a mixed C57BL/6 and C3H background [ ], and non-transgenic B6C3F1 mice (stock number: 100010) were purchased from The Jackson Laboratory.

Techniques: Western Blot, Negative Control, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of intracerebral inoculation experiments in M83 +/- mice. b ) Kaplan-Meier survival curves for M83 +/- mice inoculated with young M83 +/+ brain extract (n = 8) or with symptomatic M83 +/+ brain extract from mice exhibiting the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 22) subtype. For the symptomatic M83 +/+ inoculations, data from the three independent experiments for each subtype is pooled. Statistical significance was determined using the Log-rank test. c ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from symptomatic inoculated M83 +/- mice at the indicated DPI. One representative mouse from each of 3 replicate experiments for each M83 +/+ subtype is shown. Brain extract from an asymptomatic M83 +/- mouse inoculated with a young M83 +/+ sample is included as a negative control. Molecular weight markers indicate kDa. d ) Representative images of cortical and hippocampal (CA1 and dentate gyrus regions) PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either of the 3 different M83 +/+ subtypes. e ) Quantification of the number of cortical, CA1, and dentate gyrus PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 20) subtype. f ) Representative images of astrocytic PSyn deposits in the thalamus of symptomatic M83 +/- mice inoculated with either of 3 replicates of M83 +/+ Y brain extract. g ) Quantification of PSyn-positive astrocytes in brain sections from symptomatic M83 +/- mice inoculated with either the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 20) subtype. h ) Representative images of ring-like and Lewy body (LB)-like PSyn deposits in the midbrain and hypothalamus from symptomatic M83 +/- mice inoculated with either of the 3 M83 +/+ subtypes. i ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of symptomatic M83 +/- mice inoculated with either the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 20) disease subtype. All graphs display mean ± s.e.m. In panels e and g, statistical significance was assessed by a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. In panel i, statistical significance was assessed by two-way ANOVA. In panels d, f, and h, the scale bar indicates 20 µm and applies to all images.

Article Snippet: Homozygous M83 (M83 +/+ ) mice (stock number: 004479), which use the mouse prion protein promoter to overexpress human A53T mutant α-synuclein on a mixed C57BL/6 and C3H background [ ], and non-transgenic B6C3F1 mice (stock number: 100010) were purchased from The Jackson Laboratory.

Techniques: Western Blot, Negative Control, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of second passage experiments in M83 +/- mice. Mice were inoculated with brain extract from symptomatic M83 +/- mice that were previously inoculated with either the X (M83 +/- X ), Y (M83 +/- Y ), or Z (M83 +/- Z ) M83 +/+ subtype. b ) Kaplan-Meier survival curves for M83 +/- mice inoculated with M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain extract. Statistical significance was determined using the Log-rank test. c ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from spontaneously ill M83 +/+ mice (“passage 0”) or symptomatic M83 +/- mice at the indicated DPI following the first or second passage of the M83 +/+ X , M83 +/+ Y , or M83 +/+ Z subtypes. Molecular weight markers indicate kDa. d ) Representative images of cortical and hippocampal (CA1 and dentate gyrus regions) PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with the indicated samples. e ) Quantification of the number of cortical, CA1, and dentate gyrus PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain homogenate. f ) Representative image of astrocytic PSyn deposits in the thalamus of a symptomatic M83 +/- mouse inoculated with M83 +/- Y brain extract. g ) Quantification of PSyn-positive astrocytes in brain sections from symptomatic M83 +/- mice inoculated with either M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain homogenate. h ) Representative images of ring-like (black arrows) and Lewy body (LB)-like (red arrows) PSyn deposits in the midbrain and hypothalamus from symptomatic M83 +/- mice inoculated with the indicated samples. i ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of symptomatic M83 +/- mice inoculated with either M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain homogenate. All graphs display mean ± s.e.m. In panels e and g, statistical significance was assessed by a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. In panel i, statistical significance was assessed by two-way ANOVA. In panels d, f, and h, the scale bar indicates 20 µm and applies to all images.

Article Snippet: Homozygous M83 (M83 +/+ ) mice (stock number: 004479), which use the mouse prion protein promoter to overexpress human A53T mutant α-synuclein on a mixed C57BL/6 and C3H background [ ], and non-transgenic B6C3F1 mice (stock number: 100010) were purchased from The Jackson Laboratory.

Techniques: Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of intracerebral inoculation experiments in M83 +/- mice. b ) Kaplan-Meier survival curves for M83 +/- mice inoculated with monomeric (mono.) WT α-syn (n = 7) or with WT 1 (n = 18), WT 2 (n = 5), WT 3 (n = 8), WT 4 (n = 10), WT 5 (n = 12), or WT 6 (n = 6) PFFs. For the WT 1 and WT 5 PFF inoculations, data from two replicate experiments for each polymorph is pooled. c ) Immunoblot of detergent-insoluble PSyn species in TL-digested brain extracts from M83 +/- mice inoculated with either monomeric α-syn or WT 5 PFFs. Samples from three independent mice per inoculum are shown. Molecular weight markers indicate kDa. d ) Representative images of periventricular PSyn deposits (red arrows) in brain sections from M83 +/- mice inoculated with WT 3 or WT 6 PFFs. A brain section from a mouse inoculated with monomeric α-syn is shown as a control. e ) Quantification of the number of periventriclar PSyn deposits in brain sections from M83 +/- mice inoculated with monomeric α-syn (n = 7), WT 3 PFFs (n = 8), or WT 6 PFFs (n = 6). Statistical significance was assessed using a Kruskal-Wallis test with Dunn’s multiple comparisons test. f ) Representative images of cortical PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either of two replicates of WT 5 PFFs. g ) Quantification of the number of cortical PSyn deposits from M83 +/- mice inoculated with two independent preparations of WT 5 PFFs (n = 6 each). h ) Representative images of ring-like and Lewy body (LB)-like PSyn deposits in the midbrain of symptomatic M83 +/- mice inoculated with the indicated samples. i ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of symptomatic M83 +/- mice inoculated with either WT 1 (n = 18), WT 2 (n = 5), or WT 4 (n = 10) PFFs, or with either of two different replicates of WT 5 PFFs (n = 6 each). In panels e, g, and i, the graphs display mean ± s.e.m. In panels d, f, and h, the scale bar indicates 20 µm and applies to all images.

Article Snippet: Homozygous M83 (M83 +/+ ) mice (stock number: 004479), which use the mouse prion protein promoter to overexpress human A53T mutant α-synuclein on a mixed C57BL/6 and C3H background [ ], and non-transgenic B6C3F1 mice (stock number: 100010) were purchased from The Jackson Laboratory.

Techniques: Western Blot, Molecular Weight, Control

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of intracerebral inoculation experiments in M83 +/- mice. b ) Silver-stained SDS-PAGE of the detergent-insoluble fraction following PK digestion of monomeric (mono) recombinant A53T-mutant α-syn and the PFF samples used for inoculation experiments. c ) Kaplan-Meier survival curves for M83 +/- mice inoculated with monomeric A53T-mutant α-syn (n = 9), A53T 1 PFFs (n = 23), or A53T 2 PFFs (n = 25). For the PFF inoculations, data from the three replicate experiments for each polymorph is pooled. Statistical significance was determined using the Log-rank test. d ) Immunoblot of detergent-insoluble PSyn species in brain extracts from M83 +/- mice at the indicated DPI with either monomeric A53T α-syn (asymptomatic mice) or A53T PFFs (symptomatic mice). Two representative mice for each inoculum are shown. e ) Quantification of detergent-insoluble PSyn species in brain extracts from symptomatic M83 +/- mice inoculated with either A53T 1 PFFs (n = 18) or A53T 2 PFFs (n = 25). f ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from symptomatic M83 +/- mice at the indicated DPI with A53T 1 PFFs or A53T 2 PFFs. Two representative mice for each inoculum are shown. g ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from symptomatic M83 +/- mice at the indicated DPI with individual replicates of A53T 1 PFFs or A53T 2 PFFs. h ) Representative immunoblots for residual insoluble TL-resistant α-syn species following treatment of brain extracts from symptomatic M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs with increasing concentrations of GdnHCl. i ) [GdnHCl] 50 values for TL-resistant α-syn species in symptomatic M83 +/- mice inoculated with either A53T 1 or A53T 2 PFFs (n = 9 each, 3 mice from each replicate experiment). In panels e and i, graphs display mean ± s.e.m. and statistical significance was assessed using a Mann-Whitney test. In panels b, d, f, g, and h, molecular weight markers indicate kDa.

Article Snippet: The M83 +/+ mice used for spontaneous disease studies were either purchased from The Jackson Laboratory or bred in-house.

Techniques: Staining, SDS Page, Recombinant, Mutagenesis, Western Blot, MANN-WHITNEY, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Quantification of the area covered by PSyn staining in the indicated brain regions from symptomatic M83 +/- mice injected with A53T 1 PFFs (n = 18) or A53T 2 PFFs (n = 25). b ) Representative images of cortical PSyn deposits in brain sections from M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs. c ) Quantification of the number of cortical PSyn deposits in brain sections from M83 +/- mice inoculated with A53T 1 (n = 18) or A53T 2 (n = 25) PFFs. d ) Representative images of hippocampal PSyn deposits (CA1 and dentate gyrus regions) in brain sections from M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs. e ) Quantification of the number of PSyn deposits in the CA1 and dentate gyrus regions from M83 +/- mice inoculated with A53T 1 (n = 18) or A53T 2 (n = 25) PFFs. f ) Representative images of ring-like PSyn deposits in the midbrain and hypothalamus from M83 +/- mice inoculated with A53T 1 or A53T 2 PFFs. g ) Quantification of ring-like vs. Lewy body (LB)-like PSyn deposits in the midbrains of M83 +/- mice inoculated with A53T 1 (n = 18) or A53T 2 (n = 25) PFFs. In panels a, c, e, and g, the graphs display mean ± s.e.m. In panels a, c, and e, statistical significance was assessed by a Mann-Whitney test. In panel g, statistical significance was assessed by two-way ANOVA. In panels b, d, and f, the scale bar indicates 20 µm and applies to all images.

Article Snippet: The M83 +/+ mice used for spontaneous disease studies were either purchased from The Jackson Laboratory or bred in-house.

Techniques: Staining, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Kaplan-Meier survival curve for M83 +/+ mice (n = 32). b ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from spontaneously ill M83 +/+ mice at the indicated ages. Based on the patterns of TL- and PK-resistant species, each mouse was assigned as either subtype X, Y, or Z. Two representative mice for each subtype are shown. Brain extract from a young asymptomatic M83 +/+ mouse is included as a negative control. Molecular weight markers indicate kDa. c ) Relative frequencies of M83 +/+ X , M83 +/+ Y , and M83 +/+ Z subtypes across the 32 observed M83 +/+ mice. d ) Age of spontaneous motor dysfunction onset in M83 +/+ mice classified as M83 +/+ X (n = 8), M83 +/+ Y (n = 20), or M83 +/+ Z (n = 4). Male mice are indicated by open circles and female mice by closed circles. e ) Representative images of cortical PSyn deposits in brain sections from spontaneously sick mice exhibiting the M83 +/+ X , M83 +/+ Y , or M83 +/+ Z subtype. f ) Quantification of the number of cortical PSyn deposits in brain sections from spontaneously sick mice displaying the M83 +/+ X (n = 8), M83 +/+ Y (n = 20), or M83 +/+ Z (n = 4) subtype. g ) Representative images of ring-like and Lewy body (LB)-like PSyn deposits in the midbrain and hypothalamus from spontaneously ill M83 +/+ mice. h ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of spontaneously ill M83 +/+ mice exhibiting the M83 +/+ X (n = 8), M83 +/+ Y (n = 20), or M83 +/+ Z (n = 4) subtype. All graphs display mean ± s.e.m. In panels d and f, statistical significance was assessed by a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. In panel h, statistical significance was assessed by two-way ANOVA. In panels e and g, the scale bar indicates 20 µm and applies to all images.

Article Snippet: The M83 +/+ mice used for spontaneous disease studies were either purchased from The Jackson Laboratory or bred in-house.

Techniques: Western Blot, Negative Control, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of intracerebral inoculation experiments in M83 +/- mice. b ) Kaplan-Meier survival curves for M83 +/- mice inoculated with young M83 +/+ brain extract (n = 8) or with symptomatic M83 +/+ brain extract from mice exhibiting the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 22) subtype. For the symptomatic M83 +/+ inoculations, data from the three independent experiments for each subtype is pooled. Statistical significance was determined using the Log-rank test. c ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from symptomatic inoculated M83 +/- mice at the indicated DPI. One representative mouse from each of 3 replicate experiments for each M83 +/+ subtype is shown. Brain extract from an asymptomatic M83 +/- mouse inoculated with a young M83 +/+ sample is included as a negative control. Molecular weight markers indicate kDa. d ) Representative images of cortical and hippocampal (CA1 and dentate gyrus regions) PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either of the 3 different M83 +/+ subtypes. e ) Quantification of the number of cortical, CA1, and dentate gyrus PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 20) subtype. f ) Representative images of astrocytic PSyn deposits in the thalamus of symptomatic M83 +/- mice inoculated with either of 3 replicates of M83 +/+ Y brain extract. g ) Quantification of PSyn-positive astrocytes in brain sections from symptomatic M83 +/- mice inoculated with either the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 20) subtype. h ) Representative images of ring-like and Lewy body (LB)-like PSyn deposits in the midbrain and hypothalamus from symptomatic M83 +/- mice inoculated with either of the 3 M83 +/+ subtypes. i ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of symptomatic M83 +/- mice inoculated with either the M83 +/+ X (n = 23), M83 +/+ Y (n = 26), or M83 +/+ Z (n = 20) disease subtype. All graphs display mean ± s.e.m. In panels e and g, statistical significance was assessed by a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. In panel i, statistical significance was assessed by two-way ANOVA. In panels d, f, and h, the scale bar indicates 20 µm and applies to all images.

Article Snippet: The M83 +/+ mice used for spontaneous disease studies were either purchased from The Jackson Laboratory or bred in-house.

Techniques: Western Blot, Negative Control, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of second passage experiments in M83 +/- mice. Mice were inoculated with brain extract from symptomatic M83 +/- mice that were previously inoculated with either the X (M83 +/- X ), Y (M83 +/- Y ), or Z (M83 +/- Z ) M83 +/+ subtype. b ) Kaplan-Meier survival curves for M83 +/- mice inoculated with M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain extract. Statistical significance was determined using the Log-rank test. c ) Immunoblots of detergent-insoluble TL-resistant PSyn species and PK-resistant total α-syn species in brain extracts from spontaneously ill M83 +/+ mice (“passage 0”) or symptomatic M83 +/- mice at the indicated DPI following the first or second passage of the M83 +/+ X , M83 +/+ Y , or M83 +/+ Z subtypes. Molecular weight markers indicate kDa. d ) Representative images of cortical and hippocampal (CA1 and dentate gyrus regions) PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with the indicated samples. e ) Quantification of the number of cortical, CA1, and dentate gyrus PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain homogenate. f ) Representative image of astrocytic PSyn deposits in the thalamus of a symptomatic M83 +/- mouse inoculated with M83 +/- Y brain extract. g ) Quantification of PSyn-positive astrocytes in brain sections from symptomatic M83 +/- mice inoculated with either M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain homogenate. h ) Representative images of ring-like (black arrows) and Lewy body (LB)-like (red arrows) PSyn deposits in the midbrain and hypothalamus from symptomatic M83 +/- mice inoculated with the indicated samples. i ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of symptomatic M83 +/- mice inoculated with either M83 +/- X (n = 10), M83 +/- Y (n = 9), or M83 +/- Z (n = 8) brain homogenate. All graphs display mean ± s.e.m. In panels e and g, statistical significance was assessed by a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. In panel i, statistical significance was assessed by two-way ANOVA. In panels d, f, and h, the scale bar indicates 20 µm and applies to all images.

Article Snippet: The M83 +/+ mice used for spontaneous disease studies were either purchased from The Jackson Laboratory or bred in-house.

Techniques: Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

doi: 10.1101/2025.06.03.657690

Figure Lengend Snippet: a ) Schematic of intracerebral inoculation experiments in M83 +/- mice. b ) Kaplan-Meier survival curves for M83 +/- mice inoculated with monomeric (mono.) WT α-syn (n = 7) or with WT 1 (n = 18), WT 2 (n = 5), WT 3 (n = 8), WT 4 (n = 10), WT 5 (n = 12), or WT 6 (n = 6) PFFs. For the WT 1 and WT 5 PFF inoculations, data from two replicate experiments for each polymorph is pooled. c ) Immunoblot of detergent-insoluble PSyn species in TL-digested brain extracts from M83 +/- mice inoculated with either monomeric α-syn or WT 5 PFFs. Samples from three independent mice per inoculum are shown. Molecular weight markers indicate kDa. d ) Representative images of periventricular PSyn deposits (red arrows) in brain sections from M83 +/- mice inoculated with WT 3 or WT 6 PFFs. A brain section from a mouse inoculated with monomeric α-syn is shown as a control. e ) Quantification of the number of periventriclar PSyn deposits in brain sections from M83 +/- mice inoculated with monomeric α-syn (n = 7), WT 3 PFFs (n = 8), or WT 6 PFFs (n = 6). Statistical significance was assessed using a Kruskal-Wallis test with Dunn’s multiple comparisons test. f ) Representative images of cortical PSyn deposits in brain sections from symptomatic M83 +/- mice inoculated with either of two replicates of WT 5 PFFs. g ) Quantification of the number of cortical PSyn deposits from M83 +/- mice inoculated with two independent preparations of WT 5 PFFs (n = 6 each). h ) Representative images of ring-like and Lewy body (LB)-like PSyn deposits in the midbrain of symptomatic M83 +/- mice inoculated with the indicated samples. i ) Quantification of ring-like vs. LB-like PSyn deposits in the midbrains of symptomatic M83 +/- mice inoculated with either WT 1 (n = 18), WT 2 (n = 5), or WT 4 (n = 10) PFFs, or with either of two different replicates of WT 5 PFFs (n = 6 each). In panels e, g, and i, the graphs display mean ± s.e.m. In panels d, f, and h, the scale bar indicates 20 µm and applies to all images.

Article Snippet: The M83 +/+ mice used for spontaneous disease studies were either purchased from The Jackson Laboratory or bred in-house.

Techniques: Western Blot, Molecular Weight, Control