Phenotypes of STAM1^−/− mice. (A) Growth curves for STAM1^+/+ (male, n = 26; female, n = 9), STAM1^+/− (male, n = 36; female, n = 30), and STAM1^−/− (male, n = 21; female, n = 12) mice. One male mouse died at 7 weeks, and four male mice and two female mice died at 8 weeks. Error bars indicate standard errors. (B) Survival curves for STAM1^+/+ (male, n = 21; female, n = 31), STAM1^+/− (male, n = 27; female, n = 31), and STAM1^−/− (male, n = 21; female, n = 30) mice. (C) Abnormal limb reflex of STAM1^−/− mice. STAM1^+/+ mice extend their legs when lifted by the tail (left panel), whereas STAM1^−/− mice lack this reflex and show clasping of the hind limbs (right panel). The mice were 4-week-old littermates. (D) Priapism in STAM1^−/− mice. Priapism was observed in male STAM1^−/− mice surviving for more than 8 weeks. About 70% of STAM1^−/− male mice suffered from priapism during their lives.

Abnormalities in hippocampal CA3 subfields of STAM1^−/− mice. (A to H) HE staining of anterior coronal hippocampus sections of mice. Mice were STAM1^+/+ (A, C, E, and G) and STAM1^−/− (B, D, F, and H) and were 3 weeks old (A and B), 5 weeks old (C and D), 7 weeks old (E and F), and 9 weeks old (G and H). Note the loss of pyramidal cells in the CA3 subfields in STAM1^−/− mice (arrowheads). DG, dentate gyrus. (I to L) Anti-GFAP antibody staining. Brain sections derived from STAM1^+/+ and STAM1^−/− mice were immunostained with anti-GFAP antibody. Gliosis (arrowheads) in hippocampal CA3 subfields was observed in STAM1^−/− mice that were 4 weeks old (J) and 7 weeks old (L) but not in STAM1^+/+ mice that were 4 weeks old (I) and 7 weeks old (K). (M and N) TUNEL staining of hippocampal CA3 subfields. Hippocampus sections of 4-week-old STAM1^+/+ and STAM1^−/− mice were stained for TUNEL. Arrowheads indicate positive staining for TUNEL. (O and P) Calbindin immunostaining. The sections were prepared from the brains of 9-week-old STAM1^+/+ and STAM1^−/− mice. DG and mo, dentate gyrus and mossy fibers, respectively. Bars, 0.4 mm.

Subcellular localization of STAM1. Primary hippocampal neurons from STAM1^+/+ (A) and STAM1^−/− (B) embryos at E18.5 were immunostained with anti-STAM1 antibody after 14 days of culturing. A spot-like staining pattern (arrowheads) was observed in STAM1^+/+ neurons. Bars, 20 μm. (C to K) Coimmunostaining for STAM1 and synaptic markers. Primary hippocampal neurons from a STAM1^+/+ embryo were stained with anti-STAM1 antibody (green, C, F, and I), anti-GluR1 antibody (red, D), anti-Synapsin-I antibody (red, G), or anti-SNAP-25 antibody (red, J) or doubly stained with anti-STAM1 and each marker (E, H, and K). Considerable overlap (arrowheads) was observed between the spots of STAM1 and synaptic markers. Bars, 20 μm. (L) Subcellular fractionation of synaptosomal components prepared from mouse cerebral cortices as described in Materials and Methods. Ten micrograms of each fraction was analyzed by SDS-PAGE, electrophoretically transferred to PVDF membranes, and incubated with anti-STAM1, anti-Synapsin-I, anti-SNAP-25, and anti-PSD-95 antibodies. Syn, synaptosome.

Generation of STAM1-deficient mice. (A) Schematic representation of the mouse STAM1 (mSTAM1) cDNA, stam1 genomic locus, targeting vector, and stam1 mutated locus. The positions of stam1 exons are shown as boxes. The targeting vector was designed to replace exon 3 (E3) and E4, encoding amino acids 42 to 99 of STAM1. The fragments expected to be generated by BamHI digestion are 5.5 and 3.0 kb for the wild-type and the mutated alleles, respectively. B, BamHI; P, PstI; X, XbaI. (B) Southern blot analysis of the stam1 mutation in ES cell clones and mice. Lines indicate the positions of the DNA fragments corresponding to the wild-type (5.5 kb) and mutated (3.0 kb) alleles. (C) RT-PCR analysis of total RNA from purified splenocytes of STAM1^+/+, STAM1^+/−, and STAM1^−/− mice. The primers used are primers A and B, shown in panel A. (D) Western blot analysis for STAM1. Neocortex lysates (20 μg) from STAM1^+/+, STAM1^+/−, and STAM1^−/− mice were separated by SDS-PAGE and blotted with anti-STAM1 antibody. The position of STAM1 is marked by an arrowhead. (E) Immunoprecitipation analysis for STAM1. Lysates of activated T cells from STAM1^+/+, STAM1^+/−, and STAM1^−/− mice were immunoprecipitated and then immunoblotted with anti-STAM1 antibody. The position of STAM1 is marked by an arrowhead.

Comparison of T-cell development, proliferative responses to cytokines, and internalization of IL-2 between STAM1^+/+ and STAM1^−/− mice. (A) Flow cytometric analysis for CD4 and CD8. Thymic and splenic lymphocytes derived from 5-week-old STAM1^+/+ (n = 7) and STAM1^−/− (n = 7) mice were doubly stained with anti-CD4 and anti-CD8 MAbs. Numbers indicate the average percentages of the gated cellular subpopulations within the lymphocyte population. (B) Proliferative responses of bone marrow and spleen cells. Total splenocytes or bone marrow cells (10⁵ per well) were stimulated with the indicated ligands: 10 μg of ConA/ml, 3 μg of anti-CD3 MAb/ml, 10 nM IL-2, 100 ng of LPS/ml, 5 ng of IL-7/ml, and 30 ng of GM-CSF/ml. They were cultured for 42 h, pulsed with [³H]thymidine, and harvested after 6 h. (C) Proliferative responses of T cells. CD4⁺ T cells (10⁵ per well) purified from spleens were stimulated with anti-CD3 MAb (145.2C11) or PMA (10 ng/ml) plus ionomycin (1 μg/ml) for 42 h (left panel). ConA-activated splenic T cells (10⁵ per well) were preincubated with RPMI 1640 medium containing 1% FBS and 50 μM 2-mercaptoethanol for 12 h and then stimulated with rhIL-2 for 42 h (right panel). The cells were then pulsed with [³H]thymidine and harvested. (D) Internalization and degradation of IL-2 in activated T cells. Activated T cells from STAM1^+/+ or STAM1^−/− mice were tested for ¹²⁵I-IL-2 internalization (left panel) and degradation (right panel). Data represent means and standard errors for three independent experiments performed in triplicate.

Expression of STAM1 in adult mouse brains and embryos. (A and B) Expression of STAM1 mRNA in brains. Brain sections prepared from 4-week-old STAM1^+/+ and STAM1^−/− mice were used for in situ hybridization for STAM1 mRNA. Cb, cerebellar cortex; CP, caudate putamen; Cx, cerebral cortex; H, hippocampal formation; MO, medulla oblongata; OB, olfactory bulb; Th, thalamus. Bar, 1 mm. (C and D) Expression of STAM1 in hippocampal formation. Hippocampal sections prepared from 4-week-old STAM1^+/+ and STAM1^−/− mice were used for in situ hybridization for STAM1 mRNA. Note STAM1 mRNA in a hippocampal pyramidal cell layer from CA1 to CA3 and the dentate gyrus (DG). MH, medial habenular nucleus; CC, corpus callosum. Bar, 0.5 mm. (E and F) Expression of STAM1 mRNA in embryos. STAM1^+/+ and STAM1^−/− embryos at E18.5 were tested for STAM1 mRNA expression by in situ hybridization. BF, brown fatty tissue; Cb, cerebellar cortex; Cx, cerebral cortex; DRG, dorsal root ganglia; G, gut; H, hippocampal formation; Ht, heart; Li, liver; SP, spinal cord; Thy, thymus. Bar, 1 mm. (G and H) STAM1 immunostaining of mouse hippocampal formation at postnatal days 5 (G) and 14 (H). DG, dentate gyrus. Bars, 0.2 mm.

Susceptibility to kainic acid-induced seizures of STAM1^−/− mice. (A and B) Scores for seizures induced by kainic acid and PTZ. Four-week-old C57BL/6-STAM1^+/+ (open symbols in A) and C57BL/6-STAM1^−/− (filled symbols in A) mice were injected intraperitoneally with kainic acid (A) at doses of 10 (circles), 20 (triangles), and 30 (squares) mg per kg of body weight and with PTZ (B) at 30 and 50 mg per kg. Seizures were scored as follows: 1, arrest of motion; 2, myoclonic jerks of the head and neck with brief twitching movements; 3, unilateral clonic activity; 4, bilateral forelimb tonic and clonic activities; and 5, generalized tonic and clonic activities with loss of posture and death from continuous convulsions. At least six mice in each group were observed and scored to derive the temporal response curve. PTZ provoked rapid and abrupt general tonic-clonic convulsions, so the same criteria were used to record the PTZ-induced seizures within 5 min of injection. (C) Vulnerability of STAM1^−/− primary hippocampal neurons to cell death induced by kainic acid and an NO donor. Hippocampal neurons were isolated from C57BL/6-STAM1^+/+ and C57BL/6-STAM1^−/− embryos at E18.5. Cultured neurons were treated with 150 μM kainic acid or 50 μM SNP. After 24 h, they were examined for cell viability by the Alamar blue assay. Data represent means and standard errors for three independent experiments performed in triplicate. An asterisk indicates a P value of <0.01 for a comparison with the wild type.