Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

<div><p>Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C_18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C_18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration.</p></div

Correction: Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

<p>Correction: Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain</p

Generation of Acer3 null mouse.

<p><b>A</b>. Acer3 targeting strategy. The Acer3 gene consists of 11 exons (empty rectangles with the numerals inside). Exon 8 of the Acer3 gene was replaced by the <i>Neo</i> resistant gene cassette upon homologous recombination. <b>B</b>. Southern blot analyses of WT ES cells or ES cells from an Acer3-targeted ES clone. Genomic DNA was digested with EcoRV, resolved on a 0.8% agarose gel, transferred to a nitrocellulose membrane, which was labeled with a radioactive probe (P) corresponding to the region upstream of Exon 8 as shown in Panel A. <b>C</b>. PCR-based genotyping of Acer3^+/+, Acer3^+/-, and Acer3^-/- mice. DNA was isolated from mouse tail biopsies and subjected to PCR analyses using the PCR primer pairs (F1 and B1 or F1 and B2) as shown in Panel A. The image in C represents the PCR product patterns of the three genotypes, Acer3^+/+, Acer3^+/-, and Acer3^-/-.</p

Acer3 knockout impairs motor coordination and balance capabilities in mice.

<p><b>A</b>-<b>D</b>. Rotarod tests for motor coordination. Acer3^+/+ and Acer3^-/- mice at 6W, 4M, 6M, 8M, or 12M of age were subjected to rotarod tests under 3 task difficulties—10, 15, and 20 rpm, respectively. Hindlimb step patterns in a representative Acer3^+/+ and Acer3^-/- mouse at 8M of age at 20 rpm are displayed in D. Note that the hindpaws of Acer3^-/- mice, but not those of Acer3^+/+ mice slipped off the rod. <b>E</b>-<b>H</b>. Beam walking tests for motor coordination and balance capabilities. Acer3^+/+ and Acer3^-/- mice at 6W or 8M of age were subjected to beam walking tests under two task difficulties. The average of three trials were quantitatively analyzed for time to traverse the beam (E), walking distance (F), and foot-slips of hindpaws (G). Patterns of hindpaw contacting the beam during walking in a representative 8-month-old Acer3^+/+ and Acer3^-/- mouse are displayed in H. Note the foot-slips for both beam walking conditions in the Acer3^-/- mouse. The data in A, B, C, E, F, and G represent mean values ± SD, n = 5–8. n.s., not significant.</p

Acer3 knockout does not affect myelination.

<p><b>A</b>-<b>D</b>. Cerebellar sections of Acer3^+/+ and Acer3^-/- mice at 8M of age were stained with Luxol fast blue (A). The myelin width indicated by black arrowhead was measured in the images of the cerebellar sections stained with Luxol fast blue (B). MBP protein levels in cerebellar homogenates were analyzed by immunoblotting (C) and quantified (D). <b>E</b>. Electron microscopy of the ultrastructure of myelin sheaths in the cerebellum of Acer3^+/+ and Acer3^-/- mice at 8M of age. The image in A or E represents the results from one of four mice in each group. The data in B and D represent mean values ± SD, n = 4.</p

Acer3 upregulation is important for the homeostasis of complex sphingolipid in aging brain.

<p><b>A</b> and <b>B.</b> Levels of individual monohexosylceramide (HexCer) species and levels of total HexCers in the cerebrum (A) and cerebellum (B) of Acer3^+/+ or Acer3^-/- mice at 8M or 6W of age. Note that although causing a several-fold increase in the levels of C_18:1-HexCer in both the cerebrum and cerebellum in mice at either age, Acer3 knockout did not alter the total levels of HexCers. <b>C</b> and <b>D</b>. Levels of individual sphingomyelins and total levels of ceramides in the cerebrum (C) and cerebellum (D) of Acer3^+/+ or Acer3^-/- mice at 6W or 8Mof age. Note that Acer3 knockout only slightly increased the levels of C_18:1 or C_20:1-sphingomyelin in the cerebellum but not cerebrum in mice at either age without affecting the total levels of sphingomyelins in either brain region at either age. Total levels of sphingolipids were the sum of individual species of the same class. Data in A-D represent mean values ± SD, n = 3.</p

Acer3 knockout does not have any major defect in mouse development and fertility.

<p><b>A</b>. The average litter size of Acer3^+/+ or Acer3^-/- interbreeding mice (n = 4 litters per breeding pair) from 4 pairs of interbreeding mice. <b>B</b>. The average body weight of Acer3^+/+ or Acer3^-/- mice at 6 weeks (6W), 4 months (4M), 6 months (6M), 8 months (8M), and 12 months (12M) of age, n = 5–10 per age group. Data in A and B represent mean values ± SD.</p

Acer3 is upregulated with age in the mouse brain.

<p><b>A</b>. mRNA levels of the alkaline ceramidases Acer1, Acer2, and Acer3 in the cerebra and cerebella of young [6 weeks (6W) of age] versus middle-aged mice [8 months (8M) of age] Note that the mRNA levels of Acer3 but not Acer1 or Acer2 were increased in mice at 8M of age compared to at 6W. <b>B</b>. Acer3 mRNA levels in the cerebellum versus the cerebrum of 6W or 8M-old mice. Note that Acer3 mRNA levels were higher in the cerebellum than in the cerebrum in the same mouse at either 6W or 8M of age. <b>C</b>. Alkaline ceramidase activity on NBD-C₁₂-PHC in the brains of young (6W) versus middle-aged mice (8M). Note that the alkaline ceramidase activity was higher in both cerebrum and cerebellum in 8M-old-mice compared to 6W-old mice and that the alkaline ceramidase activity was higher in the cerebellum than in the cerebrum in mice at either age. Data in A, B, and C represent mean values ± SD, n = 3.</p

Acer3 knockout induces hindlimb clasping phenotype in mice.

<p><b>A</b> and <b>B</b>. Hindlimb clasping reflex tests. Hindlimb clasping reflex was scored in Acer3^+/+ and Acer3^-/- mice at 6W, 4M, 6M, 8M, and 12M of age (A). At 8M of age, Acer3^+/+ splayed their hindlimbs outward and away from the abdomen similar to what was seen in the 6W mice, whereas Acer3^-/- mice retracted their hindlimbs toward the abdomen into a clasping reflex (B). The data in A represent mean values ± SD, n = 5–8. n.s., not significant.</p