Retinal ganglion cell loss in kinesin-1 cargo Alcadein α deficient mice

Abstract

Maintenance of retinal ganglion cells (RGCs) activity is relied on axonal transport conveying materials required for their survival such as neurotrophic factors. Kinesin-1 undergoes anterograde transport in axons, and Alcadein α (Alcα; also called calsyntenin-1) is a major cargo adaptor protein that can drive kinesin-1 to transport vesicles containing Alcα. The long-term effects of Alcα-deficiency on retinal morphology and survival of RGCs during postnatal development were examined in Alcα knockout mice. At 1.5, 3, 6, and 15 months postnatal, the number of retrogradely labeled RGCs was determined in flat-mounted retinas of Alcα-deficient and wild-type mice. Retinal damage was assessed histologically by determining the retinal thickness. Intraocular pressure (IOP) was measured with a Tonolab tonometer. At 1.5 months postnatal, the number of retrogradely labeled RGCs was not different between wild-type and Alcα-deficient mice. However, at 3, 6, and 15 months postnatal, the number of RGCs was significantly lower in Alcα deficient mice than those of wild-type mice (143 ± 41.1 cells/mm2 vs. 208 ± 28.4 cells/mm2, respectively, at 3 months; P < 0.01). No differences were seen in retinal thickness or IOP between the two types of mice at any postnatal age. Alcα-deficient mice showed spontaneous loss of RGCs but no elevation in IOP. These mice mimic normal-tension glaucoma and will be useful for investigating the mechanism of neurodegeneration in this disorder and for developing treatments for RGC loss that does not involve changes in IOP.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Expression of Alcα in the mouse retina.

a Expression of Alcα mRNA in an eye of 5-month-old wild-type mice was detected by in situ hybridization (ISH). The magnified image in the boxed area of the left side image is shown on the right side. Alcα mRNA is readily detected in ganglion cells, cells in inner nuclear layer, and photoreceptors’ inner segment. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. Scale bar, 500 μm. b Expression of Alcα protein in an eye of 5-month-old wild-type (WT) and Alcα-deficient (KO) mouse was detected by immunohistochemistry (IHC). Alcα protein is readily detected in ganglion cells, photoreceptors’ inner segment, and outer peripheral layer. Alcα protein is not detected in the eye of Alcα-deficient mouse. Co-stained images with DAPI are shown on the right sides. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. Scale bar, 50 μm. c Expression of Alcα protein in a retina of 2-month-old wild-type (WT) and Alcα-deficient (KO) mouse was detected by western blotting. Alcα protein is not detected in the retinal lysate (1 μg) of Alcα-deficient (KO) mouse.

Fig. 2. Retrograde labeling of RGCs in wild and knockout mice.

a Typical micrographs of wild type at 15 months after birth and knockout mice at 15 months after birth. Micrographs of the central and peripheral areas were taken 0.3–0.8 mm and 1.2–1.7 mm from optic nerve head. b The number of RGCs were counted in the central (n = 8) and peripheral areas (n = 8). Result were expressed as the mean ± SEM. *P < 0.01 versus control (Student t-test). Scale bar, 100 μm.

Fig. 3. The number of RGCs of knockout mice at 1.5 (n = 8), 3 (n = 10), and 6 (n = 7) months after birth.

a In the central retina, RGC was decreased 3 months after birth. b In the peripheral retina, reduction of number of RGCs was also detected at 3 months after birth. *P < 0.05 versus control (Student t-test).

Fig. 4. Evaluating the number of RGCs with Brn-3a immunostaining.

a Typical micrographs of Brn-3a immunostaining in wild-type and knockout type at 15 months after birth. Micrographs of the central and peripheral areas were taken 0.3–0.5 mm and 1.4–1.6 mm from optic nerve head. The number of RGCs were counted with Brn-3a immunostaining in the central (b) and peripheral (c) areas. Result were expressed as the mean ± SEM. 3 M: n = 5, 8 M: n = 9, 15 M: n = 7. *P < 0.05, **P < 0.01 versus control (Student t-test). Scale bar, 50 μm.

Fig. 5. Retinal layer thickness analysis.

a Typical micrographs with hematoxylin and eosin (HE) staining in wild type and knockout type at 15 months after birth. Mean thickness of inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL). b Result were expressed as the mean ± SEM. n = 6. Scale bar, 50μm.

Fig. 6. Intraocular pressure (IOP) in wild type and knockout type.

Result were expressed as the mean ± SEM. There were no significantly differences in the IOP between wild-type and knockout mice. Wild type: broken line, knockout type: solid line. Wild type; 1.5 M: n = 10, 3 M: n = 10, 6 M: n = 20, 15 M: n = 8. Knockout type; 1.5 M: n = 8, 3 M: n = 10, 6 M: n = 6, 15 M: n = 6.