Snap29 mutant mice recapitulate neurological and ophthalmological abnormalities associated with 22q11 and CEDNIK syndrome

Abstract

Synaptosomal-associated protein 29 (SNAP29) encodes a member of the SNARE family of proteins implicated in numerous intracellular protein trafficking pathways. SNAP29 maps to the 22q11.2 region and is deleted in 90% of patients with 22q11.2 deletion syndrome (22q11.2DS). Moreover, bi-allelic SNAP29 mutations in patients are responsible for CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma) syndrome. A mouse model that recapitulates abnormalities found in these syndromes is essential for uncovering the cellular basis of these disorders. In this study, we report that mice with a loss of function mutation of Snap29 on a mixed CD1;FvB genetic background recapitulate skin abnormalities associated with CEDNIK, and also phenocopy neurological and ophthalmological abnormalities found in CEDNIK and a subset of 22q11.2DS patients. Our work also reveals an unanticipated requirement for Snap29 in male fertility and supports contribution of hemizygosity for SNAP29 to the phenotypic spectrum of abnormalities found in 22q11.2DS patients.

Keywords: Disease model; Infertility.

Fig. 1

Snap29 mRNA is ubiquitously expressed and CRISPR-mediated targeting of exon 2 depletes SNAP29 protein. a Antisense (left) and sense (right) probe of Snap29 mRNA expression at E12.5. b CRISPR design and the resulting targeted alleles, one with a 454 bp and one with a 517 bp deletion. c Western blot analysis of skin preps of mice carrying either wild-type, heterozygous, or homozygous mutant for the Snap29 allele. d Quantification of SNAP29 expression. SNAP29 levels are normalized to WT. Lb limbud, fore, or hind, l lung, hrt heart, s somite, tb tailbud. Error bar represent: standard deviation. Scale bar equals 1000 µm

Fig. 2

Snap29 mutant mice show diverse skin defects. a Example of the severe skin peeling observed in Snap29^−/− P2 pup (red arrow). b, c P3 pups showing mild skin peeling. d–e P11 pups abnormal head shape and ichtyosis on ear. f, g Example of reddish and thickened ears at P30. h–i Example of swollen reddish genetalia

Fig. 3

Snap29 mutant mice show signs of hyperkeratosis. Hematoxylin and eosin staining (a–c) and TEM (d–g) of dorsal skin. The stratum corneum (SC) in Snap29^−/− mice was found thicker and more condensed (c, e, red arrow) than that of the Snap29^+/+ or Snap29^+/− mice. Abnormal structures were seen in the extracellular spaces between granulocytes and lower corneocytes. Scale bars represent 25 µm (a–c), 2 µm (d, e), and 500 nm (f, g)

Fig. 4

Snap29 heterozygous and homozygous mutant mice have skeletal defects. Heterozygous (b) and homozygous mutant (c) embryos had extra lumbar ribs (red arrows) that were not seen in control mice (a). Advanced (d, e, yellow arrow) and delayed (f, g, black arrow) abnormal mineralization in homozygous mutant embryos

Fig. 5

Snap29 mutant female mice exhibit gait defects as measured by Catwalk. Catwalk assay was used to monitor gait parameters in Snap29^+/+, Snap29^+/−, and Snap29^−/− females. The run characteristic parameter run average was significantly slower in Snap29^−/− females (a). The temporal parameter initial dual stance was significantly elevated in both front paws (b). The spatial parameter print length was increased in both right and left front paws (c). The kinetic parameter swing speed was decreased in all paws of mutant animals (d). The interlimb coordination parameter support on two paws was significantly reduced in Snap29^−/− females (e). The grip strength was assessed for fore limbs (f) and hind limbs (g). Snap29^−/− females exhibited weaker fore and hind limbs than Snap29^+/+ animals. RF right front paw; RH right hind paw; LF left front paw, and LH left hind paw. RF and LF are in black and RH and LH are in gray. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars represent standard error of the Mean

Fig. 6

Snap29 mutant male mice exhibit fewer gait defects as measured by Catwalk. Catwalk assay was used to monitor gait parameters in Snap29^+/+, Snap29^+/−, and Snap29^−/− males. The kinetic parameter swing speed was decreased on the left side of Snap29^−/− males (a). The interlimb coordination parameter support on two paws was significantly reduced in Snap29^−/− males (b). The grip strength was assessed for fore limbs (c) and hind limbs (d). Snap29^−/− males exhibited weaker fore and hind limbs than Snap29^+/+ animals. RF right front paw; RH right hind paw; LF left front paw; and LH left hind paw. RF and LF are in black and RH and LH are in gray. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars represent Standard error of the Mean

Fig. 7

Analysis of ophthalmological defects in Snap29^−/− mice. Representative scotopic (a–c; amplitude (in microvolt) and time (in millisecond) calibrations at the left of tracing a) and photopic (d–f; amplitude (in microvolt) and time (in millisecond) calibrations at the left of tracing d) ERG wave forms. a Scotopic ERG recorded from wild-type mouse; b Scotopic ERG recorded from heterozygous mouse; c Scotopic ERG recorded from homozygous mouse with low b wave amplitude; d photopic ERG recorded from wild-type mouse; e photopic ERG recorded from heterozygous mouse; f photopic ERG recorded from homozygous mouse with low b wave amplitude. In tracings a and d, the ERG a- and b-waves are identified with letters a and b, respectively. Representative eye histology of wild-type male mouse (g) at P118; heterozygous female at P83 (h) and homozygous female mouse at P87(i). ISL inner segment layer, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer. Scale bar represents 25 µm

Fig. 8

Histological analysis of Snap29^−/− testis. Testis sections were stained with hematoxylin and eosin to evaluate spermatogenesis in WT (a, c) and Snap29^−/− (b, d) mice. WT testis displayed normal spermatogenesis (Stages VII and VIII showing elongating spermatids and spermatozoa in the lumen). Seminiferous tubules in Snap29^−/− testis had degenerated germ cells (white arrows), loss of immature germ cells accumulated in the lumen (arrow heads), giant multinucleated spermatids (black arrow) and extensive vacuolization (*). The diameter of degenerated seminiferous tubules was reduced in Snap29^−/− (d) compared with WT (c) testis