Screening for synapsin isoforms that allow α-syn functionality.

A) Schematic showing pH-sensitive sensor sypHy and principle of pHluorin experiments to quantitatively evaluate the SV cycle (see main text and methods for more details).

B) Elimination of all synapsins block α-syn functionality at synapses. Left: Schematic showing design of pHluorin experiments. WT or synapsin TKO cultured hippocampal neurons were co-transduced at 5 days in-vitro (DIV) with h-α-syn:mCherry (or mCherry as control) and sypHy, and imaged at 14-15 DIV. Right: Stimulation-induced sypHy fluorescence traces (300 action potentials at 20 Hz, delivered at t=0 sec – for clarity, symbols only mark every other mean±SEM ΔF/F0 value in all sypHy traces). Note that while h-α-syn over-expression (orange) attenuated sypHy fluorescence in WT neurons, there was no effect in neurons from mice lacking all synapsins (TKO). All sypHy data quantified in (C).

C) Quantification of peak ΔF/F0 sypHy values. A total of 12-19 coverslips were analyzed for each condition, from at least 3 separate cultures (*** p=1e-7, ns p=0.90, U-test).

D) Domain structure of the five main synapsin isoforms.

E) Experimental design to identify the synapsin isoform that reinstated α-syn functionality, Synapsin TKO neurons were co-transduced at 5 DIV with each synapsin isoform, h-α-syn, and sypHy; and imaged at 14-15 DIV.

F) SypHy fluorescence traces (mean±SEM). Note that h-α-syn (orange) attenuates SV recycling only if the neurons are also co-expressing the “a” isoforms – synapsins Ia, IIa and IIIa (300 action potentials at 20 Hz, delivered at t=0 sec). Data quantified in G.

G) Quantification of peak ΔF/F0 sypHy values. 13-26 coverslips from at least 3 separate cultures were analyzed for each condition (***p=0.0009, ns p=0.62, *** p=0.00005, ns p=0.62 for Ib and p=0.99 for IIb; ** p=0.004, Student’s t test).

Interaction of synapsin isoforms with h-α-syn.

A) Workflow for co-immunoprecipitation experiments in neuro2a cells.

B) Western blots from co-immunoprecipitation experiments show that the synapsin isoforms Ia, IIa, and IIIa associate more robustly with h-α-syn (top panel), when compared to synapsins Ib and IIb (a non-specific band is marked with an asterisk).

C) Quantification of blots in (B) n=5, all data presented as mean ± SEM (** p < 0.01, *** p < 0.001, Student’s t-test).

D) Schematic showing synapsin isoforms and their variable domains. Note that the E-domain is common between synapsins Ia, IIa and IIa.

E) Workflow for pulldown of GST-tagged h-α-syn WT/deletions/scrambled mutations after incubation with mouse brain lysates. Equivalent amounts of immobilized GST α-syn variants were used.

F) Schematic showing α-syn regions that were scrambled (amino acids between 96-140 and 96-110).

G) Top: Samples from GST-pulldown were analyzed by NuPAGE and immunoblotted with an antibody against synapsin I (top panel). Bottom: Ponceau staining shows equivalent loading of fusion proteins. Note that full-length h-α-syn bound synapsin I from mouse brains (lane 2), while deletion of the h-α-syn C-terminus (amino acids 96-140, lane 3) eliminated this interaction. Lanes 4-7 show that the sequence within amino acids 96-110 of h-α-syn is critical for binding to synapsin I. All western blots are quantified below (n=3). Data presented as mean ± SEM *** p < 0.001, Student’s t-test.

The synapsin E-domain is necessary and sufficient for enabling α-syn functionality.

A) Schematic showing synapsin Ia scrambled E-domain sequence (synapsin Iascr-E). Numbers depict amino acid positions, letters in the inset depict amino-acids. Note that the WT amino acids are randomized in the scrambled mutant.

B) Design of sypHy experiments co-expressing synapsin Iascr-E and h-α-syn in cultured neurons from synapsin TKO mice.

C) Stimulation-induced sypHy fluorescence traces (300 action potentials at 20 Hz, delivered at t=0 sec). Note that while h-α-syn attenuated sypHy fluorescence in synapsin TKO neurons expressing synapsin Ia, h-α-syn had no effect in neurons expressing synapsin Iascr-E. Insets: Quantification of peak ΔF/F0 sypHy values. 10-15 coverslips from at least 3 separate cultures were analyzed for each condition (** ; p=0.0057, one-way ANOVA with Tukey’s posthoc analysis).

D) Top: Schematic for co-immunoprecipitation experiments, to test the interaction of h-α-syn with WT synapsin Ia or synapsin Iascr-E. Neuro2a cells were co-transfected with myc-tagged α-syn and respective YFP-tagged synapsin Ia, and the YFP was immunoprecipitated. Bottom: Note that h-α-syn co-immunoprecipitated with synapsin Ia, but not synapsin Iascr-E; quantification of the gels below (n=4, all data are means ± SEM *** p < 0.001, Student’s t test – a non-specific band is marked with an asterisk).

E) Schematic of experiments to test if the synapsin E-domain is sufficient to enable α-syn functionality in synapsin TKO neurons. Synapsin-E (a 46 amino acid sequence) was fused to the C-terminus of sypHy, so that upon expression in neurons, the E-domain would be present on the cytosolic surface of SVs.

F) SypHy fluorescence traces. Note that while h-α-syn (orange) was unable to attenuate SV recycling in synapsin TKO neurons (as expected), diminished synaptic responses were seen when the E-domain was present. Insets: Quantification of peak ΔF/F0 sypHy values. 12-19 coverslips from at least 3 separate cultures were analyzed for each condition (***p=1.2e-7, one-way ANOVA with Tukey’s posthoc analysis).