Figure 1.

PRRT2 Is Localized at the Presynaptic Level

(A) Ultrafractionation of brain synaptosomes. Purified synaptosomes from adult mouse brain were subjected to ultrasynaptic fractionation to separate the AZ, PSD, and NSSP made by extrinsic and integral membrane proteins of the nerve terminal. Aliquots of total synaptosomes and of each ultrasynaptic fraction (10–30 μg) were probed with antibodies against PRRT2, SNAP-25, and protein markers to validate ultrasynaptic compartments such as synaptophysin-1 (Syp1) and PSD95 (top). Immunoblots were quantified by densitometric analysis of the fluorograms, and the values are expressed in mean (± SEM) percentages of the total amount (bottom). The partition of PSD95, Syp1, and SNAP-25 in the corresponding fractions is shown. Note that PRRT2 preferentially partitioned in the NSSP fraction, similarly to Syp1 and SNAP-25.

(B) Subcellular distribution of endogenous PRRT2 in neurons. Forebrain fractions obtained at various stages of SV purification were analyzed by western blotting using antibodies to PRRT2, SNAP-25, and Syp1 (top). H, homogenate; S1, post-nuclear supernatant; S2, supernatant of P2; P2, crude synaptosomes; LP1, crude synaptic plasma membranes; LS1, supernatant of LP1; LP2, crude synaptic vesicles; LS2, synaptosol; USV, highly purified synaptic vesicles; SSV, salt-treated highly purified synaptic vesicles; FT, flowthrough fraction containing small presynaptic membranes. Immunoblots were quantified as in (A), and the value of each subcellular fraction is expressed in percentage of homogenate as means ± SEM (bottom).

(C) Localization of PRRT2 in mature neurons. Primary hippocampal neurons transduced at 10 DIVs with PRRT2-mCherry (red) were subjected to immunostaining at 15 DIVs with antibodies to PRRT2, SNAP-25, and Syp1. PRRT2 immunoreactivity (red) largely overlapped with the staining of the two presynaptic proteins in axonal and nerve terminal areas. Scale bar, 10 μm.

See also Figure S1.