Anat Cell Biol.  2014 Jun;47(2):101-110. 10.5115/acb.2014.47.2.101.

Region-specific changes in the immunoreactivity of Atg9A in the central nervous system of SOD1(G93A) transgenic mice

• Lee JC
• Choe SY
• Cha CI

• KMID: 1882580
• DOI: http://doi.org/10.5115/acb.2014.47.2.101

Abstract

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.

Keyword

Amyotrophic lateral sclerosis; SOD1(G93A) transgenic mice; Atg9A; Cerebral cortex; Hippocampus; Thalamus

MeSH Terms

Amyotrophic Lateral Sclerosis
Animals
Autophagy
Brain
Central Nervous System*
Cerebellum
Cerebral Cortex
Hippocampus
Homeostasis
Mice
Mice, Transgenic*
Spinal Cord
Superoxide Dismutase
Thalamus
Tissue Distribution
Superoxide Dismutase

Figure

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  Fig. 1 Localizations of Atg9A-immunoreactive cells in the cerebral cortex of 18w wtSOD1 transgenic (A, C) and 18w symptomatic SOD1(G93A) transgenic mice (B, D). Panels (C) and (D) are high power views of panels (A) and (B), respectively. In the cerebral cortex of SOD1(G93A) transgenic mice (B, D), intensely stained Atg9A positive cells are seen in layers II-VI of the parietal association cortex, compared with the same areas in wtSOD1 transgenic mice (A, C). At a higher magnification, Atg9A-immunoreactive cells in symptomatic SOD1(G93A) transgenic mice illustrated the typical morphology of cell bodies (D), whereas weakly stained cell bodies were found in wtSOD1 transgenic mice (C). Scale bars=150 µm (A, B), 30 µm (C, D).

• 

  Fig. 2 Localization of Atg9A-immunoreactive cells in the hippocampus of 18w wtSOD1 transgenic (A, B, E, G) and 18w symptomatic SOD1(G93A) transgenic mice (C, D, F, H). There were layer-specific alterations in the number of Atg9A-immunoreactive cells in the hippocampus (A, C). Panels (B) and (D) are high power views of the CA1 regions of panels (A) and (C), respectively. It was noted that the pyramidal cell layers in CA1 region were strongly immunoreactive for Atg9A in symptomatic SOD1(G93A) transgenic mice (D). Panels (E) and (F) are high power views of the CA3 regions and dentate gyrus of panels (G) and (H), respectively. In symptomatic SOD1(G93A) transgenic mice, Atg9A immunoreactivity was significantly increased in all three layers of the CA3 areas (F), and the alteration was prominent in the pyramidal cell layers. In the dentate gyrus, Atg9A immunoreactivity was also increased in the granule cell layers and polymorphic layers in symptomatic SOD1(G93A) transgenic mice (H). CA1-3, fields CA1-3 of Ammon's horn; DGlb, dentate gyrus, lateral blade; DGmb, dentate gyrus, medial blade; G, granule cell layer; O, stratum oriens; P, pyramidal cell layer; Po, polymorphic layer, R, stratum radiatum. Scale bars=200 µm (A, C), 30 µm (B, D, E-H).

• 

  Fig. 3 Localization of Atg9A-immunoreactive cells in the thalamus of 18w wtSOD1 transgenic (A, C, E, G) and 18w symptomatic SOD1(G93A) transgenic mice (B, D, F H). Insets in panels (A), (B), (C), and (D) indicate the areas magnified in panels (E), (F), (G), and (H). Panels (E) and (F) is the same region of the control mice as panels (G) and (H). DLG, dorsal lateral geniculate nucleus; VLG, ventral lateral geniculate nucleus; LPMR, lateral posterior thalamus nucleus, mediorostral; LPLR, lateral posterior thalamus nucleus, laterorostral; IMA, intramedullary thalamus area. Scale bars=100 µm (A-D), 20 µm (E-H).

• 

  Fig. 4 Localization of Atg9A-immunoreactive cells in the cerebellum of 18w wtSOD1 transgenic (A, B) and 18w symptomatic SOD1(G93A) transgenic mice (C, D). Panels (B) and (D) are high power views of panels (A) and (C), respectively. In the cerebellar cortex of wtSOD1 transgenic mice, intensely stained Atg9A-positive cells were seen in granular layer, molecular layer, and Purkinje cell layer (A, B), while Atg9A positive cells were strongly detected in the same layer in SOD1(G93A) transgenic mice (C, D). G, granular layer; M, molecular layer; Pu, Purkinje cell layer; W, white matter. Scale bars= 50 µm (A, C), 20 µm (B, D).

• 

  Fig. 5 Localization of Atg9A-immunoreactive cells in the spinal cord of 18w wtSOD1 transgenic (A, C) and 18w symptomatic SOD1(G93A) transgenic mice (B, D). (C) and (D) are high power views of (A) and (B), respectively. In the spinal cord of SOD1(G93A) transgenic mice (B, D), intensely stained Atg9A-positive cells were seen in the spinal cord layers, dorsal cortiscospinal tract and anterior horn of lumbar segments, in contrast with the same areas in wtSOD1 transgenic mice (A, C). AH, anterior horn; cc, central canal; dcs, dorsal cortiscospinal tract; gr, gracile fasciculus. Scale bars=200 µm (A, B), 30 µm (C, D).

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