Differential functions of the Apoer2 intracellular domain in selenium uptake and cell signaling

Abstract

Apolipoprotein E receptor 2 (Apoer2) is a multifunctional transport and signaling receptor that regulates the uptake of selenium into the mouse brain and testis through endocytosis of selenoprotein P (Sepp1). Mice deficient in Apoer2 or Sepp1 are infertile, with kinked and hypomotile spermatozoa. They also develop severe neurological defects on a low selenium diet, due to a profound impairment of selenium uptake. Little is known about the function of Apoer2 in the testis beyond its role as a Sepp1 receptor. By contrast, in the brain, Apoer2 is an essential component of the Reelin signaling pathway, which is required for proper neuronal organization and synapse function. Using knock-in mice, we have functionally dissociated the signaling motifs in the Apoer2 cytoplasmic domain from Sepp1 uptake. Selenium concentration of brain and testis was normal in the knock-in mutants, in contrast to Apoer2 knock-outs. Thus, the neurological defects in the signaling impaired knock-in mice are not caused by a selenium uptake defect, but instead are a direct consequence of a disruption of the Reelin signal. Reduced sperm motility was observed in some of the knock-in mice, indicating a novel signaling role for Apoer2 in sperm development and function that is independent of selenium uptake.

Figure 1. Apoer2 ICD mutant knockin mice

Apoer2[ex19] mice constitutively express the full length receptor containing the alternatively spliced exon 19. Apoer2[Δex19] mice constitutively express the full length receptor without the alternatively spliced exon 19. Apoer2[Stop] mice express a truncated receptor lacking the C-terminal ICD after the NPXY domain. Apoer2[Dab^-] Mice express the full length receptor with a mutation in the Dab-1 binding site.

Figure 2. No significant difference in tissue selenium levels of Apoer2 ICD mutant mice from wild type

Tissue selenium values were measured from mice of all six genotypes after four weeks on a 0.25 mg/kg selenium diet. Only Apoer2 KO mice presented with significantly lower selenium levels than wild type mice in the brain (A) and testes (B); **P<0.001. Liver selenium levels were comparable among all genotypes (C).

Figure 3. Vesicular Sepp1 staining observed in all Apoer2 ICD mutant mice

Fixed mouse testes were stained for Sepp1. Vesicular Sepp1 staining (arrows) is apparent in WT mice and all Apoer2 ICD mutants. As previously reported, there is no Sepp1 vesicular staining in Apoer2 KO testes. Scale bar = 10 μm.

Figure 4. Sperm immunostaining reveals normal organization of spermatozoa in all Apoer2 mutant mice

Fixed spermatozoa were immunostained with MitoFluor™ (midpiece; red), a crossreacting polyclonal antibody against an unidentified sperm antigen (acrosome and principal piece; green), and DAPI (nucleus; blue). (A) wild type (B) Apoer2[ex19] (C) Apoer2[Δex19] (D) Apoer2[Stop] (E) Apoer2[Dab^-] (F) Apoer2 KO. Pictures are representative of the major morphological sperm population for each genotype. Arrows indicate location of annulus. Scale bar = 10 μm.

Figure 5. Sperm classification into four morphological categories

The majority of spermatozoa observed fell into one of four different morphological categories: straight, hairpin (sperm tail is bent back on itself at the annulus), angled (sperm tail is slightly bent at the annulus creating an angled tail), and curled (the tail is curled proximal to the head of the spermatozoon). Asterisks mark sperm head. Arrow marks annulus