Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system

Abstract

The synucleins are a unique family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human neurodegenerative diseases. We have cloned a new member of the synuclein family called persyn. In contrast to other synucleins, which are presynaptic proteins of CNS neurons, persyn is a cytosolic protein that is expressed predominantly in the cell bodies and axons of primary sensory neurons, sympathetic neurons, and motoneurons. Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry revealed that persyn mRNA and protein are expressed in these neurons from the earliest stages of axonal outgrowth and are maintained at a high level throughout life. Persyn also becomes detectable in evolutionary recent regions of the brain by adulthood.

Fig. 1.

Persyn transcripts in mouse and rat tissues. Northern hybridization of 5–20 μg of total RNA extracted from various tissues of E13 mouse embryos (a) and newborn rat (b) with a nick-translatedpersyn probe. After stripping off the probe, the same filter was hybridized with a nick-translated cDNA fragment encoding the mouse L27 ribosomal protein to provide an indication of the amount of total RNA from each tissue present on the filter. TG, Trigeminal ganglia; DRG, dorsal root ganglia.

Fig. 2.

Homologies between mouse persyn and other members of the synuclein family. Alignment of persyn and other members of the synuclein family. Amino acids identical in all or in all but one members of the family are shown in white on black background. Gaps (-) were introduced for better alignment. Sequence accession numbers: Torpedo californica synuclein, P37379; rat synucleins 1, 2, 3, products of alternative splicing of the same geneS73007, S73008, S73009; human α-synuclein (synuclein 1/NACP), L36674; human β-synuclein (synuclein 2), S69965; bovine synuclein, P33567; zebrafinch synelfin, L33860; rat synuclein-like, X86789; human BCSG1,AF010126; mouse persyn, AF017255; human persyn, AF017256.

Fig. 3.

Top Left. Whole-mount in situhybridization of mouse embryos. E11 mouse embryos were hybridized with a DIG-labeled antisense cRNA persyn probe.TG, Trigeminal ganglion; VG, vestibular ganglion; GG, geniculate ganglion; JG, jugular ganglion; NG, nodose ganglion;DRG, dorsal root ganglia; MN, motoneurons in ventral horns of the spinal cord.

Fig. 7.

Developmental time course of persyn expression in the trigeminal ganglia. a, Graph showing the changes in the level of persyn mRNA in the trigeminal ganglia during development. The mean ± SEM of the persyn mRNA levels relative to GAPDH mRNA levels are shown (n = 3).b, Western blot showing the developmental changes in the level of persyn protein the trigeminal ganglia. Equal amounts (15 μg) of total protein from embryonic (E) or newborn (P0) mouse trigeminal ganglia were run in each lane.

Fig. 8.

Specificity of anti-mouse persyn antibody. Western blot/ECL detection of persyn protein with affinity-purified SK-23 antibody. a, b, Equal amounts (15 μg) of total protein from P0 mouse heart and hindbrain and E13 and E17 trigeminal ganglia were run in each lane. Western blots were probed with SK-23 antibody (a) or SK-23 antibody preincubated with recombinant mouse persyn protein (b) as described in Materials and Methods. c, Fifteen micrograms of total protein from newborn (P0) mouse trigeminal ganglia (the tissue with a high level of persyn expression) and cerebral cortex (the tissue where persyn expression could not be detected but with substantial levels of α- and β-synucleins expression) and 0.5 μg of recombinant α- and β-synucleins were analyzed.

Fig. 9.

Persyn in subcellular fractions from mouse spinal cord. Western blot showing the distribution of persyn protein in subcellular fractions of the adult mouse spinal cord.hmg, Homogenate; cyt, 1000 gm supernatant; pmt, 14,000 gm supernatant;pmc, 120,000 gm supernatant; mcs, 120,000 gm pellet; msk, 14,000 gm pellet; mt, mitochondrial fraction; syn, synaptosomal fraction. Equal amounts (15 μg) of total protein from each fraction were run in each lane. Very similar results were obtained with subcellular fractions of trigeminal ganglia, midbrain, and hindbrain.