Differential expression of SNAP‐25 family proteins in the mouse brain

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25 is a neuronal SNARE protein essential for neurotransmitter release from presynaptic terminals. Three palmitoylated SNAP-25 family proteins: SNAP-25a, SNAP-25b, and SNAP-23, are expressed in the brain, but little is known about their distributions and functions. In the present study, we generated specific antibodies to distinguish these three homologous proteins. Immunoblot and immunohistochemical analyses revealed that SNAP-25b was distributed in synapse-enriched regions throughout almost the entire brain, whereas SNAP-25a and SNAP-23 were expressed in relatively specific brain regions with partially complementary expression patterns. SNAP-25a and SNAP-25b, but not SNAP-23, were also present in the axoplasm of nerve fibers. The intracellular localization was also different, and although SNAP-25b and SNAP-23 were found primarily in membrane and lipid raft-enriched fractions of mouse brain homogenates, a substantial amount of SNAP-25a was recovered in soluble fractions. In PC12 cells, SNAP-25b was localized to the plasma membrane, but SNAP-25a and SNAP-23 were distributed throughout the cytoplasm. The expression and distribution of these three proteins were also differentially regulated in the early postnatal period. These results indicate that the three SNAP-25 family proteins display a differential distribution in the brain as well as in neuronal cells, and possibly play distinct roles.

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

“…We identified SNAP-25B-specific peptides in all samples, but specific SNAP-25A peptides only in a few. This indicates that SNAP-25B levels were much higher than SNAP-25A levels, consistent with earlier findings that SNAP-25B is the predominant species in adult brain (18,28). Overall, the identified tryptic peptides of SNAP-25B covered the whole protein (amino acids 2-198, supplemental Fig.…”

“…Because they are both strongly associated into complexes and membrane associated, the SNARE proteins are difficult to analyze via mass spectrometry, which is incompatible with most detergents necessary for the solubilization of proteins. Each SNARE complex protein exists in several isoforms that are differently distributed within the central nervous system (13)(14)(15)(16)(17)(18). Posttranslational modifications and truncated variants of the SNARE proteins make investigation of the protein expression even more complicated.…”

Accumulation of α-Synuclein Triggered by Presynaptic Dysfunction

“…Coronal sections of the striatum and the entire rostrocaudal extent of the substantia nigra (SN) were cut serially at 20 m thickness using a cryostat (CM1850; Leica Microsystems). For Western blotting and catecholamine measurement by HPLC, brain blocks of mice perfused transcardially with PBS were dissected using the method described previously by our group, to yield the striatal tissues and ventral parts of midbrain tissues including the SN (Yasuda et al, 2011). After completing the dissection, the sections were immediately frozen on dry ice and stored at Ϫ80°C until analysis.…”

“…The primary antibodies used for confocal microscopy were mouse anti-␣Syn (clone 42; diluted at 1:100; BD Biosciences) (TapiaGonzález et al, 2011), rabbit anti-␣Syn (1:500; Millipore Bioscience Research Reagents) (Chung et al, 2003;Herzig et al, 2011), rabbit antityrosine hydroxylase (TH) (1:5000; Calbiochem) (Yasuda et al, 2011), mouse anti-synaptophysin (clone SY38; 1:500; Millipore) (Masliah et al, 2001;Tabuchi et al, 2007;Gimbel et al, 2010), rat anti-dopamine transporter (DAT) (clone MAB369; 1:500; Millipore) (Herzig et al, 2011;Kaushal et al, 2011), rabbit or goat anti-vesicular glutamate transporter-1 (VGLUT1) (1:300; Frontier Institute) (Miyazaki et al, 2003;Kawamura et al, 2006) , mouse anti-syntaxin 1 (10H5) (1.4 g/ml) , rabbit anti-VAMP-2 (1.4 g/ml) , mouse anti-synaptophysin (clone SY38; 1:500; Millipore), rabbit anti-TH (1:500; Calbiochem), and rabbit anti-␤-tubulin (1:500; Abcam). For immunoelectron microscopy, the primary antibodies used were mouse anti-␣Syn (clone 42; 1 g/ml; BD Biosciences), guinea pig anti-DAT (1 g/ml; Frontier Institute), and rabbit anti-VGLUT1 (1 g/ml; Frontier Institute).…”

“…For DA cell counting in the SN pars compacta (SNpc), every fourth 20-m-thick serial section of the brain was immunostained for TH and counterstained with cresyl violet (Nissl staining) (Furuya et al, 2004;Yasuda et al, 2011). The numbers of TH-and Nissl-doublepositive cells in the SNpc were counted both in wild-type and Snap25 S187A/S187A mice in a blind manner.…”