Fluorescence normalized high resolution melting curves of wild type (red), ob/ob (green), and ob/+ (blue) animals.

<p>Fluorescence normalized high resolution melting curves of wild type (red), ob/ob (green), and ob/+ (blue) animals.</p

Ob forward and reverse primer sequences for HRM.

<p>Ob forward and reverse primer sequences for HRM.</p

Confirmation of HRM results by alternate gel-based methods.

<p>Locus specific control band at 191^st lane and Lep^ob specifc band at 123bp in 2^nd lane of pairs.</p

Weights of WT and Lep^ob animals from our breeding colonies genotyped by HRM method.

<p>Weights of WT and Lep^ob animals from our breeding colonies genotyped by HRM method.</p

A simplified schematic of the HRM protocol.

<p>A simplified schematic of the HRM protocol.</p

Primers and product around Lep^ob SNP that yields premature stop codon.

<p>Primers and product around Lep^ob SNP that yields premature stop codon.</p

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

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

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