Amelioration of enteric neuropathology in a mouse model of Niemann-Pick C by Npc1 expression in enteric glia

Abstract

Niemann-Pick C (NPC) disease is an autosomal recessive, lethal, neurodegenerative disorder caused by mutations in NPC1. By using the glial fibrillary acidic protein (GFAP) promoter, we demonstrated previously that astrocyte-specific expression of Npc1 decreased neuronal storage of cholesterol in Npc1(-/-) mice; reduced numbers of axonal spheroids; and produced less degeneration of neurons, reactive astrocytes, and loss of myelin tracts in the central nervous system. GFAP-Npc1, Npc1(-/-) mice exhibited markedly enhanced survival, and death was not associated with the severe terminal weight loss observed in Npc1(-/-) mice. Intestinal transit is delayed in Npc1(-/-) mice but is normal in GFAP-NPC1, Npc1(-/-) until late in the course of their disease. Because glia play an important role in the enteric nervous system, we studied morphology and cholesterol content of intestines from Npc1(-/-) mice and examined the effect of GFAP-promoted restoration of Npc1 in enteric glia. Although the number of neurons was not altered, the total amount of cholesterol stored in the small intestine was decreased, as were the number of neurons with inclusions and the number of inclusions per neuron. We conclude that expression of Npc1 by enteric glial cells can ameliorate the enteric neuropathology, and we speculate that dysfunction of the enteric nervous system contributes to the retarded intestinal transit, weight loss, and demise of Npc1(-/-) mice.

Figure 1

Weights of GFAP-Npc1, Npc1^−/− (diamonds), Npc1^−/− mice (asterisks), and Npc1^+/+ (circles) mice. The error bars provide standard deviations. (Modified from Zhang et al., 2008 with additional data.)

Figure 2

Western blot analysis of Npc1 expression in large intestine. The diffuse band at 180 kDa is caused by glycosylation of Npc1. β-actin is used as a loading control.

Figure 3

Light microscopic appearance of H&E-stained and anti-Hu immunostained sections of colon from Npc1^+/− (A-C), Npc1^−/− (D-F), and GFAP-Npc1, Npc1^−/− (G-I). Low magnification images (A, D, G) of the gut wall and higher magnification of myenteric ganglia illustrate a similar distribution of myenteric (arrowheads) and submucosal (arrows) ganglia in the three genotypes. Foamy macrophages were present focally in the submucosa of Npc1^−/− (m in panel D) and GFAP-Npc1, Npc1^−/− (not shown), as opposed to Npc1^+/− controls (A). In Npc1^−/− samples (D, E), and to a lesser degree in GFAP-NPC1, Npc1^−/− mice (G, H), neuronal cell bodies (n) and nerves in both plexuses, as well as intermuscular nerves (i) appear vacuolated and enlarged. In contrast, glia (g) do not appear affected in any of the three genotypes. Hu immunoreactivity identifies the perikarya of myenteric neurons, which are enlarged and vacuolated in Npc1^−/− samples, in contrast with the other genotypes. Scale bars = 20 μm.

Figure 4

Ultrastructural appearance of myenteric ganglia in Npc1^+/− (A, B), Npc1^−/− (C, D), and GFAP-Npc1, Npc1^−/− (E, F) mice. (A) Normal features of myenteric ganglia include intimately associated neurons (n) and glial cells (g) with mitochondrial, endoplasmic reticulum, cytoskeletal filaments, and other organelles, but no pathological inclusions. (B) The filament-rich cytoplasm of an enteric glial cell (g) appears more electron dense than a neuronal cell body (n) or neuronal processes (arrowheads). The glial cell extends multiple processes (asterisks), which surround portions of the neuronal cell body and interdigitate between axons and dendrites. (C) The majority of neuronal perkarya (n) in Npc1^−/− mice are swollen with cytoplasmic membranous inclusions, but glial cells (g) are not affected. (D) Higher magnification of cytoplasmic inclusions demonstrates their lamellar appearance. (E) Enteric glia (g) and many neurons (n) in GFAP-Npc1, Npc1^−/− mice do not contain inclusions. Those neurons in which inclusions persist (nx) have fewer vacuoles than in Npc1^−/− mice. In this example, their perikarya appear “shrunken” and electron dense with condensed nuclei, but this was not a consistent observation. (F) Higher magnification of perikaryal cytoplasm from a neuron with no inclusions. (m) = smooth muscle. Scale bars = 2 microns.

Figure 5

Micrograms of cholesterol per mg protein in Npc1^+/+ (white); Npc1^−/− (grey); and GFAP-Npc1, Npc1^−/− (black) mice. Error bars are std. dev. (a) small intestine (*p ≤ 0.05, **p ≤ 0.01 to Npc1^−/−; Student T Test). (b) large intestine (*p ≤ 0.05 to Npc1^+/+; Student T Test).